Influenza hemagglutinin antibodies, compositions and related methods

ABSTRACT

Antibodies against influenza hemagglutinin, compositions containing the antibodies, and methods of using the antibodies are provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority from U.S. ProvisionalApplication Ser. No. 61/246,958, filed on Sep. 29, 2009.

TECHNICAL FIELD

This invention relates to influenza hemagglutinin antibodies, and tomaterials and methods for making and using influenza hemagglutininantibodies.

BACKGROUND

Influenza has a long history characterized by waves of pandemics,epidemics, resurgences and outbreaks. Influenza is a highly contagiousdisease that could be equally devastating both in developing anddeveloped countries. The influenza virus presents one of the majorthreats to the human population. In spite of annual vaccination efforts,influenza infections result in substantial morbidity and mortality.Although flu epidemics occur nearly every year, fortunately pandemics donot occur very often. However, recent flu strains have emerged such thatwe are again faced with the potential of an influenza pandemic. Avianinfluenza virus of the type H5N1, currently causing an epidemic inpoultry in Asia as well as regions of Eastern Europe, has persistentlyspread throughout the globe. The rapid spread of infection, as well ascross species transmission from birds to human subjects, increases thepotential for outbreaks in human populations and the risk of a pandemic.The virus is highly pathogenic, resulting in a mortality rate of overfifty percent in birds as well as the few human cases which have beenidentified. If the virus were to achieve human to human transmission, itwould have the potential to result in rapid, widespread illness andmortality.

Subtypes of the influenza virus are designated by different HA and NAresulting from antigenic shift. Furthermore, new strains of the samesubtype result from antigenic drift, or mutations in the HA or NAmolecules which generate new and different epitopes. While technologicaladvances have improved the ability to produce improved influenzaantigens vaccine compositions, there remains a need to provideadditional sources of protection against to address emerging subtypesand strains of influenza.

SUMMARY

This document relates to antibody compositions and methods for producingantibody compositions, including production in plant systems. Thisdocument further relates to vectors encoding antibodies or antigenbinding fragments thereof, as well as fusion proteins, plant cells,plants, compositions, and kits comprising antibodies or antigen bindingfragments thereof, and therapeutic and diagnostic uses in associationwith influenza infection in a subject.

This document is based in part on the identification of anti-H5N1hemagglutinin monoclonal antibodies (mAbs) that specifically inhibithemagglutination of highly pathogenic avian influenza (HPAI). Theprotective efficacy of one of these antibodies has been demonstrated inanimal challenge models (e.g., mouse models) using homologous virus. Thespecific and effective inhibition of these antibodies makes them usefulas therapeutic tool in the treatment and/or prevention of humaninfection. In addition, the mAbs can be a useful diagnostic tool fortyping suspected H5N1 human isolates in conjunction with otherdiagnostic approaches. Thus, this document provides antibodies againstinfluenza hemagglutinin antigens, as well as antibody componentsproduced in plants. The antibodies can inhibit hemagglutination. Alsoprovided are antibody compositions that are reactive against influenzahemagglutinin antigen. In addition, methods for production and use ofthe antibodies and compositions are provided herein.

Thus, in a first aspect, this document features an isolated monoclonalantibody that binds hemagglutinin, wherein the antibody has the abilityto inhibit hemagglutination, and wherein the antibody is selected fromthe group consisting of an antibody comprising a light chain variableregion amino acid sequence at least 85% identical to the amino acidsequence as set forth in amino acids 1-97 of SEQ ID NO:79 and a heavychain variable region amino acid sequence at least 85% identical to theamino acid sequence as set forth in amino acids 1-115 of SEQ ID NO:78;and an antibody comprising a light chain variable region amino acidsequence at least 85% identical to the amino acid sequence as set forthin amino acids 1-96 of SEQ ID NO:81 and a heavy chain variable regionamino acid sequence at least 85% identical to the amino acid sequence asset forth in amino acids 1-112 of SEQ ID NO:80.

The antibody can have a light chain variable region amino acid sequenceat least 90% identical to the amino acid sequence as set forth in aminoacids 1-97 of SEQ ID NO:79, and a heavy chain variable region amino acidsequence at least 90% identical to the amino acid sequence as set forthin amino acids 1-115 of SEQ ID NO:78. The antibody can have a lightchain variable region amino acid sequence at least 95% identical to theamino acid sequence as set forth in amino acids 1-97 of SEQ ID NO:79,and a heavy chain variable region amino acid sequence at least 95%identical to the amino acid sequence as set forth in amino acids 1-115of SEQ ID NO:78. The antibody can have a light chain variable regionamino acid sequence at least 98% identical to the amino acid sequence asset forth in amino acids 1-97 of SEQ ID NO:79, and a heavy chainvariable region amino acid sequence at least 98% identical to the aminoacid sequence as set forth in amino acids 1-115 of SEQ ID NO:78. Theantibody can have a light chain variable region amino acid sequence atleast 99% identical to the amino acid sequence as set forth in aminoacids 1-97 of SEQ ID NO:79, and a heavy chain variable region amino acidsequence at least 99% identical to the amino acid sequence as set forthin amino acids 1-115 of SEQ ID NO:78. The antibody can have a lightchain variable region amino as set forth in amino acids 1-97 of SEQ IDNO:79, and a heavy chain variable region amino acid sequence as setforth in amino acids 1-115 of SEQ ID NO:78.

The antibody can have a light chain variable region amino acid sequenceat least 90% identical to the amino acid sequence as set forth in aminoacids 1-96 of SEQ ID NO:81, and a heavy chain variable region amino acidsequence at least 90% identical to the amino acid sequence as set forthin amino acids 1-112 of SEQ ID NO:80. The antibody can have a lightchain variable region amino acid sequence at least 95% identical to theamino acid sequence as set forth in amino acids 1-96 of SEQ ID NO:81,and a heavy chain variable region amino acid sequence at least 95%identical to the amino acid sequence as set forth in amino acids 1-112of SEQ ID NO:80. The antibody can have a light chain variable regionamino acid sequence at least 98% identical to the amino acid sequence asset forth in amino acids 1-96 of SEQ ID NO:81, and a heavy chainvariable region amino acid sequence at least 98% identical to the aminoacid sequence as set forth in amino acids 1-112 of SEQ ID NO:80. Theantibody can have a light chain variable region amino acid sequence atleast 99% identical to the amino acid sequence as set forth in aminoacids 1-96 of SEQ ID NO:81, and a heavy chain variable region amino acidsequence at least 99% identical to the amino acid sequence as set forthin amino acids 1-112 of SEQ ID NO:80. The antibody can have a lightchain variable region amino as set forth in amino acids 1-96 of SEQ IDNO:81, and a heavy chain variable region amino acid sequence as setforth in amino acids 1-112 of SEQ ID NO:80.

In another aspect, this document features an antibody that bindshemagglutinin, wherein the antibody has the ability to inhibithemagglutination, and wherein the antibody is selected from the groupconsisting of an antibody comprising a light chain amino acid sequenceat least 85 percent identical to the amino acid sequence set forth inSEQ ID NO:79 and a heavy chain amino acid sequence at least 85 percentidentical to the amino acid sequence set forth in SEQ ID NO:78; and anantibody comprising a light chain amino acid sequence at least 85percent identical to the amino acid sequence set forth in SEQ ID NO:81and a heavy chain amino acid sequence at least 85 percent identical tothe amino acid sequence set forth in SEQ ID NO:80.

The antibody can have a light chain amino acid sequence at least 90%identical to the amino acid sequence set forth in SEQ ID NO:79 and aheavy chain amino acid sequence at least 90% identical to the amino acidsequence set forth in SEQ ID NO:78. The antibody can have a light chainamino acid sequence at least 95% identical to the amino acid sequenceset forth in SEQ ID NO:79, and a heavy chain amino acid sequence atleast 95% identical to the amino acid sequence set forth in SEQ IDNO:78. The antibody can have a light chain amino acid sequence at least98% identical to the amino acid sequence set forth in SEQ ID NO:79 and aheavy chain amino acid sequence at least 98% identical to the amino acidsequence set forth in SEQ ID NO:78. The antibody can have a light chainamino acid sequence at least 99% identical to the amino acid sequenceset forth in SEQ ID NO:79 and a heavy chain amino acid sequence at least99% identical to the amino acid sequence set forth in SEQ ID NO:78. Theantibody can have a light chain amino acid sequence as set forth in SEQID NO:79 and a heavy chain amino acid sequence as set forth in SEQ IDNO:78.

The antibody can have a light chain amino acid sequence at least 90%identical to the amino acid sequence set forth in SEQ ID NO:81 and aheavy chain amino acid sequence at least 90% identical to the amino acidsequence set forth in SEQ ID NO:80. The antibody can have a light chainamino acid sequence at least 95% identical to the amino acid sequenceset forth in SEQ ID NO:81 and a heavy chain amino acid sequence at least95% identical to the amino acid sequence set forth in SEQ ID NO:80. Theantibody can have a light chain amino acid sequence at least 98%identical to the amino acid sequence set forth in SEQ ID NO:81 and aheavy chain amino acid sequence at least 98% identical to the amino acidsequence set forth in SEQ ID NO:80. The antibody can have a light chainamino acid sequence at least 99% identical to the amino acid sequenceset forth in SEQ ID NO:81 and a heavy chain amino acid sequence at least99% identical to the amino acid sequence set forth in SEQ ID NO:80. Theantibody can have a light chain amino acid sequence as set forth in SEQID NO:81 and a heavy chain amino acid sequence as set forth in SEQ IDNO:80.

Any of the antibodies featured herein can be an scFv, Fv, Fab′, Fab,diabody, linear antibody or F(ab′)2 antigen-binding fragment of anantibody; a CDR, univalent fragment, or a single domain antibody; ahuman, humanized or part-human antibody or antigen-binding fragmentthereof, or a recombinant antibody. The antibody can be produced in aplant.

Any of the antibodies featured herein can be operatively attached to abiological agent or a diagnostic agent. For example, an antibody can beoperatively attached to an agent that cleaves a substantially inactiveprodrug to release a substantially active drug. The drug can be ananti-influenza agent. An antibody can be operatively attached to ananti-viral agent (e.g., an anti-influenza agent). An antibody can beoperatively attached to a biological agent as a fusion protein preparedby expressing a recombinant vector that comprises, in the same readingframe, a DNA segment encoding the antibody operatively linked to a DNAsegment encoding the biological agent. An antibody can be operativelyattached to a biological agent via a biologically releasable bond orselectively cleavable linker.

An antibody can be operatively attached to a diagnostic, imaging ordetectable agent. For example, an antibody can be operatively attachedto an X-ray detectable compound, a radioactive ion or a nuclear magneticspin-resonance isotope. An antibody can be operatively attached to (a)the X-ray detectable compound bismuth (III), gold (III), lanthanum (III)or lead (II); (b) the detectable radioactive ion copper67, gallium67,gallium68, indium111, indium113, iodine-123, iodine-125, iodine-131,mercury197, mercury203, rhenium186, rhenium188, rubidium97, rubidium103,technetium99m or yttrium90; or (c) the detectable nuclear magneticspin-resonance isotope cobalt (II), copper (II), chromium (III),dysprosium (III), erbium (III), gadolinium (III), holmium (III), iron(II), iron (III), manganese (II), neodymium (III), nickel (II), samarium(III), terbium (III), vanadium (II) or ytterbium (III). An antibody canbe operatively attached to biotin, avidin or to an enzyme that generatesa colored product upon contact with a chromogenic substrate.

In another aspect, this document features a nucleic acid comprising anucleotide sequence encoding an antibody light chain or an antibodyheavy chain as provided herein. An expression vector containing thenucleic acid also is provided. The expression vector can further includea nucleotide sequence encoding a leader sequence.

This document also features a host cell containing an expression vectoras provided herein. The host cell can be a plant cell.

In addition, this document features a plant comprising a plant cell asprovided herein. The plant can be from a genus selected from the groupconsisting of Brassica, Nicotiana, Petunia, Lycopersicon, Solanum,Capsium, Daucus, Apium, Lactuca, Sinapis, or Arabidopsis. The plant canbe from a species selected from the group consisting of Nicotianabenthamiana, Brassica carinata, Brassica juncea, Brassica napus,Brassica nigra, Brassica oleraceae, Brassica tournifortii, Sinapis alba,and Raphanus sativus. The plant can be selected from the groupconsisting of alfalfa, radish, mustard, mung bean, broccoli, watercress,soybean, wheat, sunflower, cabbage, clover, petunia, tomato, potato,tobacco, spinach, and lentil. The plant can be a sprouted seedling.

In another aspect, this document features a recombinant, plant-producedmonoclonal antibody that binds hemagglutinin, wherein the antibody hasthe ability to inhibit hemagglutination, and wherein the antibody isselected from the group consisting of an antibody comprising a lightchain amino acid sequence at least 85 percent identical to the aminoacid sequence set forth in SEQ ID NO:79 and a heavy chain amino acidsequence at least 85 percent identical to the amino acid sequence setforth in SEQ ID NO:78; and an antibody comprising a light chain aminoacid sequence at least 85 percent identical to the amino acid sequenceset forth in SEQ ID NO:81 and a heavy chain amino acid sequence at least85 percent identical to the amino acid sequence set forth in SEQ IDNO:80.

The recombinant, plant-produced monoclonal antibody can have a lightchain amino acid sequence at least 95% identical to the amino acidsequence set forth in SEQ ID NO:79, and a heavy chain amino acidsequence at least 95% identical to the amino acid sequence set forth inSEQ ID NO:78. The recombinant, plant-produced monoclonal antibody canhave a light chain amino acid sequence at least 98% identical to theamino acid sequence set forth in SEQ ID NO:79 and a heavy chain aminoacid sequence at least 98% identical to the amino acid sequence setforth in SEQ ID NO:78. The recombinant, plant-produced monoclonalantibody can have a light chain amino acid sequence at least 99%identical to the amino acid sequence set forth in SEQ ID NO:79 and aheavy chain amino acid sequence at least 99% identical to the amino acidsequence set forth in SEQ ID NO:78. The recombinant, plant-producedmonoclonal antibody can have a light chain amino acid sequence as setforth in SEQ ID NO:79 and a heavy chain amino acid sequence as set forthin SEQ ID NO:78.

The recombinant, plant-produced monoclonal antibody can have a lightchain amino acid sequence at least 90% identical to the amino acidsequence set forth in SEQ ID NO:81 and a heavy chain amino acid sequenceat least 90% identical to the amino acid sequence set forth in SEQ IDNO:80. The recombinant, plant-produced monoclonal antibody can have alight chain amino acid sequence at least 95% identical to the amino acidsequence set forth in SEQ ID NO:81 and a heavy chain amino acid sequenceat least 95% identical to the amino acid sequence set forth in SEQ IDNO:80. The recombinant, plant-produced monoclonal antibody can have alight chain amino acid sequence at least 98% identical to the amino acidsequence set forth in SEQ ID NO:81 and a heavy chain amino acid sequenceat least 98% identical to the amino acid sequence set forth in SEQ IDNO:80. The recombinant, plant-produced monoclonal antibody can have alight chain amino acid sequence at least 99% identical to the amino acidsequence set forth in SEQ ID NO:81 and a heavy chain amino acid sequenceat least 99% identical to the amino acid sequence set forth in SEQ IDNO:80. The recombinant, plant-produced monoclonal antibody can have alight chain amino acid sequence as set forth in SEQ ID NO:81 and a heavychain amino acid sequence as set forth in SEQ ID NO:80.

In still another aspect, this document features a pharmaceuticalcomposition comprising an antibody as provided herein, and apharmaceutically acceptable carrier. The composition can be formulatedfor parenteral or topical administration. The antibody can be arecombinant, plant-produced antibody. The pharmaceutically acceptablecomposition can be an encapsulated or liposomal formulation. Thecomposition can further comprise a second therapeutic agent.

This document also features use of a composition as provided herein fortreating an influenza infection in a subject in need thereof, as well asuse of a composition as provided herein in the manufacture of amedicament for treating an influenza infection.

In another aspect, this document features a method for determiningwhether a subject is at risk for influenza virus infection. The methodcan include contacting a biological sample from the subject with anantibody as provided herein. The subject can be a human.

In yet another aspect, this document features a method for typing aninfluenza virus, comprising contacting the influenza virus with anantibody as provided herein, and if binding of the antibody to theinfluenza virus is detected, typing the influenza virus as an H5 virus.

This document also features a method for treating a subject in needthereof, comprising contacting a biological sample from the subject withan antibody as provided herein and, if the antibody shows detectablebinding to the biological sample, administering an antibody as providedherein to the subject. The subject can be a human. The subject can bediagnosed as having influenza.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the plant viral vector pGRD4-H5 HA.

FIG. 2A is a graph depicting the activity of mAb 4F5 against homologousand heterologous strains of influenza viruses. FIG. 2B is a graphdepicting the activity of mAb 5F5 against homologous and heterologousstrains of influenza viruses. FIG. 2C is a graph depicting the activityof mAb 1E11 against homologous and heterologous strains of influenzaviruses.

FIG. 3 is a table summarizing hemagglutinin inhibition activity ofanti-HA mAbs.

FIG. 4 is a table summarizing binding activity of anti-HA mAbs.

FIG. 5 is a table summarizing hemagglutination inhibition activity ofanti-H5 HA mAbs.

FIG. 6 is a table summarizing hemagglutination inhibition activity ofanti-H3 HA mAbs.

FIG. 7 depicts the experimental design used to evaluate the protectiveefficacy of mAbs in mice.

FIG. 8 depicts the results of an experiment to evaluate the protectiveefficacy of mAbs in mice.

FIG. 9 depicts the amino acid sequences, including signal peptidesequences, of heavy and light chains for mAbs 1E11 and 4F5.

FIG. 10 depicts the amino acid sequences, without signal peptidesequences, of heavy and light chains for mAbs 1E11 and 4F5.

DETAILED DESCRIPTION

This document relates to influenza antibodies that can be useful toprevent, delay onset of, treat, ameliorate symptoms of, reduceoccurrence of, and/or diagnose influenza infection. This document alsorelates to antibody compositions, and methods of production of providedantibody compositions, including but not limited to, production in plantsystems. Further, this document relates to vectors, fusion proteins,plant cells, plants and compositions comprising antibodies or antigenbinding fragments thereof. Still further provided are kits as well astherapeutic and diagnostic uses in association with influenza infectionin a subject.

Influenza Antigens

In general, influenza antigens can include any immunogenic polypeptidethat elicits an immune response against influenza virus. Immunogenicpolypeptides of interest can be provided as independent polypeptides, asfusion proteins, as modified polypeptides [e.g., containing additionalpendant groups such as carbohydrate groups, alkyl groups (such as methylgroups, ethyl groups, or propyl groups), phosphate groups, lipid groups,amide groups, formyl groups, biotinyl groups, heme groups, hydroxylgroups, iodo groups, isoprenyl groups, myristoyl groups, flavin groups,palmitoyl groups, sulfate groups, or polyethylene glycol]. In someembodiments, influenza antigen polypeptides for use in accordance withthis disclosure have an amino acid sequence that is or includes asequence identical to that of an influenza polypeptide found in nature;in some embodiments influenza antigen polypeptides have an amino acidsequence that is or includes a sequence identical to a characteristicportion (e.g., an immunogenic portion) of an influenza polypeptide foundin nature.

In certain embodiments, full length proteins are utilized as influenzaantigen polypeptides in vaccine compositions in accordance with thisdisclosure. In some embodiments, one or more immunogenic portions ofinfluenza polypeptides are used. In certain embodiments, two or three ormore immunogenic portions are utilized, as one or more separatepolypeptides or linked together in one or more fusion polypeptides.

Influenza antigen polypeptides can include, for example, full-lengthinfluenza polypeptides, fusions thereof, and/or immunogenic portionsthereof. Where portions of influenza proteins are utilized, whetheralone or in fusion proteins, such portions retain immunological activity(e.g., cross-reactivity with anti-influenza antibodies). Based on theircapacity to induce immunoprotective response against viral infection,hemagglutinin is an antigen of interest in generating vaccines.

In certain embodiments, full length hemagglutinin (HA) is utilized togenerate HA antibodies as provided herein. In some embodiments one ormore domains of HA can be used. In certain embodiments, two or three ormore domains are utilized, as one or more separate polypeptides orlinked together in one or more fusion polypeptides. Sequences ofexemplary HA polypeptides are presented in Table 1.

TABLE 1 Exemplary HA Sequences GenBank Accession Strain HA SequenceABY51347 A/environment/ 5′MNIQILAFIACVLTGAKGDKICLGHHAVANGTKVNTLTEKGI NewEVVNATETVETADVKKICTQGKRATDLGRCGLLGTLIGPPQCD York/3181-QFLEFSSDLIIERREGTDVCYPGRFTNEESLRQILRRSGGIGKES 1/2006MGFTYSGIRTNGAASACTRSGSSFYAEMKWLLSNSDNSAFPQ (H7N2)MTKAYRNPRNKPALIIWGVHHSESASEQTKLYGSGNKLITVRSSKYQQSFTPSPGTRRIDFHWLLLDPNDTVTFTFNGAFIAPDRASFFRGESLGVQSDAPLDSSCRGDCFHSGGTIVSSLPFQNINSRTVGRCPRYVKQKSLLLATGMRNVPEKPKPRGLFGAIAGFIENGWEGLINGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIGKTNQQFELIDNEFNEIEQQIGNVINWTRDAMTEIWSYNAELLVAMENQHTIDLADSEMSKLYERVKKQLRENAEEDGTGCFEIFHKCDDQCMESIRNNTYDHTQYRTESLQNRIQIDPVKLSSGYKDIILWFSFGASCFILLAIAMGLVFICIKNGNMQCTICI 3′ (SEQ ID NO: 1) ACC61810A/environment/ 5′MNTQILAFIACVLTGVKGDKICLGHHAVANGTKVNTLTEKG NewIEVVNATETVETADVKKICTQGKRATDLGRCGLLGTLIGPPQC York/3185-DQFLEFSSDLIIERREGTDVCYPGRFTNEESLRQILRRSGGIGKE 1/2006SMGFTYSGIRTNGATSACTRSGSSFYAEMKWLLSNSDNSAFPQ (H7N2)MTKAYRNPRNKPALIIWGVHHSESVSEQTKLYGSGNKLITVRSSKYQQSFTPSPGARRIDFHWLLLDPNDTVTFTFNGAFIAPDRASFFRGESLGVQSDVPLDSSCRGDCFHSGGTIVSSLPFQNINSRTVGKCPRYVKQKSLLLATGMRNVPEKPKPRGLFGAIAGFIENGWEGLINGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIGKTNQQFELIDNEFNEIEQQIGNVINWTRDAMTEIWSYNAELLVAMENQHTIDLADSEMSKLYERVKKQLRENAEEDGTGCFEIFHKCDDQCMESIRNNTYDHTQYRTESLQNRIQIDPVKLSSGYKDIILWFSFGASCFLLLAIAMGLVFICIKNGNMQCTICI 3′ (SEQ ID NO: 2) ABI26075A/guineafowl/ 5′MNIQILAFIACVLTGAKGDKICLGHHAVANGTKVNTLTEKGI NY/4649-EVVNATETVETANIKKICTQGKRPTDLGQCGLLGTLIGPPQCD 18/2006QFLEFSSDLIIERREGTDVCYPGKFTNEESLRQILRRSGGIGKES (H7N2)MGFTYSGIRTNGATSACTRSGSSFYAEMKWLLSNSDNAAFPQMTKSYRNPRNKPALIIWGVHHSESVSEQTKLYGSGNKLIKVRSSKYQQSFTPNPGARRIDFHWLLLDPNDTVTFTFNGAFIAPDRASFFRGESIGVQSDAPLDSSCGGNCFHNGGTIVSSLPFQNINPRTVGKCPRYVKQKSLLLATGMRNVPEKPKKRGLFGAIAGFIENGWEGLINGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIGKTNQQFELINNEFNEVEQQIGNVINWTQDAMTEVWSYNAELLVAMENQHTIDLTDSEMSKLYERVRKQLRENAEEDGTGCFEIFHKCDDHCMESIRNNTYDHTQYRTESLQNRIQIDPVKLSGGYKDIILWFSFGASCFLLLAIAMGLVFICIKNGNMQCTICI 3′ (SEQ ID NO: 3) ABR37506A/environment/ 5′MNTQILALIAYMLIGAKGDKICLGHHAVANGTKVNTLTERG Maryland/267/IEVVNATETVETVNIKKICTQGKRPTDLGQCGLLGTLIGPPQCD 2006(H7N3)QFLEFDADLIIERREGTDVCYPGKFTNEESLRQILRGSGGIDKESMGFTYSGIRTNGVTSACRRSGSSFYAEMKWLLSNSDNAAFPQMTKSYRNPRNKPALIIWGVHHSGSATEQTKLYGSGNKLITVGSSKYQQSFTPSPGARPQVNGQSGRIDFHWLLLDPNDTVTFTFNGAFIAPDRASFFRGESLGVQSDVPLDSGCEGDCFHSRGTIVSSLPFQNINPRTVGKCPRYVKQTSLLLATGMRNVPENPKTRGLFGAIAGFIENGWEGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIDKTNQQFELIDNEFSEIEQQIGNVINWTRDSMTEVWSYNAELLVAMENQHTIDLADSEMNKLYERVRKQLRENAEEDGTGCFEIFHKCDDQCMESIRNNTYDHTQYRTESLQNRIQIDPVKLSSGYKDIILWFSFGASCFLLLAIAMGLVFICIKNGNMRCTI CI 3′ (SEQ ID NO: 4)ACF47475 A/mallard/ 5′MNTQILALIACMLIGAKGDKICLGHHAVANGTKVNTLTERGCalifornia/ IEVVNATETVETANIKKICTQGKRPTDLGQCGLLGTLIGPPQCD HKWFQFLEFDADLIIERREGTDVCYPGKFTNEESLRQILRGSGGIDKES 1971/2007MGFTYSGIRTNGATSACRRSGSSFYAEMKWLLSNSDNAAFPQ (H7N7)MTKSYRNPRNKPALIIWGVHHSGSATEQTKLYGSGNKLITVGSSKYQQSFTPSPGARPQVNGQSGRIDFHWLLLDPNDTVTFTFNGAFIAPDRASFFRGGSLGVQSDVPLDSGCEGDCFHSGGTIVSSLPFQNINPRTVGKCPRYVKQTSLLLATGMRNVPENPKTRGLFGAIAGFIENGWEGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIDKTNQQFELIDNEFNEIEQQIGNVINWTRDSMTEVWSYNAELLVAMENQHTIDLADSEMNKLYERVRKQLRENAEEDGTGCFEIFHKCDDQCMESIRNNTYDHTQYRTESLQNRIQINPVKLSSGYKDIILWFSFGASCFLLLAIAMGLVFICIKNGNMRCTI CI 3′ (SEQ ID NO: 5)ABP96852 A/Egypt/2616- 5′MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVNAMRU3/2007 THAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCD (H5N1)EFLNVPEWSYIVEKINPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSDYEASSGVSSACPYQGRSSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQIRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKSNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIINKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESMGIYQILSIYSTVASSLALAIMVAGLFLW MCSNGSLQCRICI 3′(SEQ ID NO: 6) ABV23934 A/Nigeria/6e/5′DQICIGYHANNSTEQVDTIMEKNVTVTHAQNILEKTHNGKL 07(H5N1)CDLDGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKIVKKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKIRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLTLAIMVAGLSLWMCSNGSLQCRICI 3′ (SEQ ID NO: 7) ABI16504A/China/GD01/ 5′MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTV 2006THAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCD (H5N1)EFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIISKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNNAAEQTKLYQNPTTYISVGTSTLNLRLVPKIATRSKVNGQSGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPLRERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLW MCSNGSLQCRICI 3′(SEQ ID NO: 8) ABY27653 A/India/m777/5′MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTV 2007 (H5N1)THAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKTSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRETRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSN GSLQCRIC 3′ (SEQ ID NO: 9)AB136046 A/Indonesia/ 5′DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDC326N/2006 CDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKA (H5N1)NPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILNPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMMAGLSLWMCSNGSLQCRICI 3′ (SEQ ID NO: 10) ACD85624A/Mississippi/ 5′MKTIIALSYILCLVSAQKFPGNDNSTATLCLGHHAVPNGTIV 05/2008KTITNDQIEVTNATELVQSSSTGEICDSPHQILDGENCTLIDALL (H3N2)GDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEEFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIRSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGN IRCNICI 3′ (SEQ ID NO: 11)ACF10321 A/New 5′MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIV York/06/2008KTITNDQIEVTNATELVQSSSTGEICDSPHQILDGENCTLIDALL (H3N2)GDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNKEKFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRLRVRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGN IRCNICI 3′ (SEQ ID NO: 12)ACD85628 A/Idaho/03/ 5′MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIV2008 (H3N2) KTITNDQIEVTNATELVQSSSTGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSKCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGN IRCNICI 3′ (SEQ ID NO: 13)ACF40065 A/Louisiana/ 5′MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIV06/2008 KTITNDQIEVTNATELVQSSSTGEICDSPHQILDGENCTLIDALL (H3N2)GDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGN IRCNICI 3′ (SEQ ID NO: 14)ACB11768 A/Indiana/01/ 5′MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNV2008 (H1N1) TVTHSVNLLENSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYHWTLLEPGDTIIFEANGNLIAPRYAFTLSRGFGSGIINSNAPMDKCDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWM CSNGSLQCRICI 3′(SEQ ID NO: 15) ACB11769 A/Pennsylvania/5′MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNV 02/2008TVTHSVNLLENSHNGKLCLLKGIAPLQLGNCSVAGWILGNPEC (H1N1)ELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKTLYHTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNAPMDKCDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMC SNGSLQCRICI 3′(SEQ ID NO: 16) ACD47238 A/Alaska/02/5′MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNV 2008 (H1N1)TVTHSVNLLENSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKESAWPNHTVTGVSASCSHNGEXSFYRNLLWLTXKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHTENAYVSVVSSHYSRKFTPEIAKRPKVRXQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNAPMDKCDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNXKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTXDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWM CSNGSLQCRICI 3′(SEQ ID NO: 17) ACD85766 A/Indiana/04/5′MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNV 2008 (H1N1)TVTHSVNLLENNHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEMFPKEGSWPNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLXKSYANNKEKEVLVLWGVHHPPNIGDQKALYHTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNAPMDNCDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVXKEFNKLERRMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISF WMCSNGSLQCRICI 3′(SEQ ID NO: 18) ACF40125 A/Wisconsin/5′MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNV 01/2008TVTHSVNLLENSHNGKLCLLKGIAPLQLGNCSVAGWILGNPEC (H1N1)ELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPDIGDQKTLYHTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNAPMDKCDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMC SNGSLQCRICI 3′(SEQ ID NO: 19) Vietnam 5′AKAGVQSVKMEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDH5N1 TIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLAL ALMVAGLSLWMCSNGSLQCRICI 3′(SEQ ID NO: 20) Wyoming 5′MKTIIALSYILCLVFSQKLPGNDNSTATLCLGHHAVPNGTIVH3N2 KTITNDQIEVTNATELVQSSSTGGICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWAGVTQNGTSSACKRRSNKSFFSRLNWLTHLKYKYPALNVTMPNNEKFDKLYIWGVHHPVTDSDQISLYAQASGRITVSTKRSQQTVIPNIGYRPRVRDISSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGN IRCNICI 3′ (SEQ ID NO: 21)DQ371928 A/Anhui/1/2005 5′MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTV(H5N1) THAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPLRERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLW MCSNGSLQCRICI 3′(SEQ ID NO: 22) ISDN125873 A/Indonesia/5/5′MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTV 05THAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMMAGLSLWM CSNGSLQCRICI 3′(SEQ ID NO: 23) DQ137873 A/bar-headed5′MERIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTV goose/Qinghai/THAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCD 0510/05EFLNVPEWSYIVEKINPANDLCYPGNFNDYEELKHLLSRINHFE (H5N1)RIQIIPKSSWSDHEASSGVSSACPYQGRSSFFRNVVWLIKKNNAYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPENAYKNCQKGDSTIMKSELEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQGERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLS LWMCSNG 3′ (SEQ ID NO: 24)A/VietNam/ 5′MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTV 1194/04THAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSL WMCSNGSLQCRICI 3′(SEQ ID NO: 25) B/Brisbane/3/5′MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVT 07GVIPLTTTPTKSYFANLKGTKTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLANLLRGYENIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKSGFFATMAWAVPKDNNKNATNPLTVEVPYICTEGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGGFPDQTEDGGLPQSGRIVVDYMMQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMLAIFIVYMVSRDNVSCSICL 3′ (SEQ ID NO: 26) ACA28844A/Brisbane/59/ 5′MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNV 2007 (H1N1)TVTHSVNLLENSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNAPMDKCDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWM CSNGSLQCRICI 3′(SEQ ID NO: 27) A/Brisbane/10/5′QKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATEL 2007 (H3N2)VQSSSTGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFPYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNI 3′ (SEQ ID NO: 28) ACA33493B/Florida/4/ 5′MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVT 2006GVIPLTTTPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVKPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKSGFFATMAWAVPKDNNKNATNPLTVEVPYICTEGEDQITVWGFHSDDKTQMKNLYGDSNPQKFTSSANGVTTHYVSQIGSFPDQTEDGGLPQSGRIVVDYMMQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMLAIFIVYMVSRDNVSCSICL 3′ (SEQ ID NO: 29) B/Malaysia/5′MKAIIVLLMVVTSNADRIICTGITSSNSPHVVKTATQGEVNV 2506/2004-likeTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGNIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNSLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFVVYMVSRDNVSCSICL 3′ (SEQ ID NO: 30) AAP34324 A/New5′MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNV Caledonia/20/TVTHSVNLLEDSHNGKLCLLKGIAPLQLGNCSVAGWILGNPEC 99 (H1N1)ELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDECDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNNECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWM CSNGSLQCRICI 3′(SEQ ID NO: 31) ABU99109 A/Solomon5′MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNV Islands/3/2006TVTHSVNLLEDSHNGKLCLLKGIAPLQLGNCSVAGWILGNPEC (H1N1)ELLISRESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKESSWPNHTTTGVSASCSHNGESSFYKNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQRALYHKENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNAPMDECDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWM CSNGSLQCRICI 3′(SEQ ID NO: 32) A/Wisconsin/67/5′MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIV 2005KTITNDQIEVTNATELVQSSSTGGICDSPHQILDGENCTLIDALL (H3N2)GDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNDESFNWTGVTQNGTSSSCKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPVTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAINQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIR CNICI 3′ (SEQ ID NO: 33)AAT08000 A/Wyoming/3/ 5′MKTIIALSYILCLVFSQKLPGNDNSTATLCLGHHAVPNGTIV03(H3N2) KTITNDQIEVTNATELVQSSSTGGICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWAGVTQNGTSSACKRRSNKSFFSRLNWLTHLKYKYPALNVTMPNNEKFDKLYIWGVHHPVTDSDQISLYAQASGRITVSTKRSQQTVIPNIGYRPRVRDISSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGN IRCNICI 3′ (SEQ ID NO: 34)AAR02640 A/Netherlands/ 5′SKSRGYKMNTQILVFALVASIPTNADKICLGHHAVSNGTKV219/03 NTLTERGVEVVNATETVERTNVPRICSKGKRTVDLGQCGLLG (H7N7)TITGPPQCDQFLEFSADLIIERREGSDVCYPGKFVNEEALRQILRESGGIDKETMGFTYSGIRTNGTTSACRRSGSSFYAEMKWLLSNTDNAAFPQMTKSYKNTRKDPALIIWGIHHSGSTTEQTKLYGSGNKLITVGSSNYQQSFVPSPGARPQVNGQSGRIDFHWLILNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSEVQVDANCEGDCYHSGGTIISNLPFQNINSRAVGKCPRYVKQESLLLATGMKNVPEIPKRRRRGLFGAIAGFIENGWEGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIEKTNQQFELIDNEFTEVERQIGNVINWTRDSMTEVWSYNAELLVAMENQHTIDLADSEMNKLYERVKRQLRENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHSKYREEAIQNRIQIDPVKLSSGYKDVILWFSFGASCFILLAIAMGLVFICV KNGNMRCTICI 3′(SEQ ID NO: 35)

While sequences of exemplary influenza antigen polypeptides are providedherein, it will be appreciated that any sequence having immunogeniccharacteristics of HA may be employed. In some embodiments, an influenzaantigen polypeptide can have an amino acid sequence that is about 60%identical, about 70% identical, about 80% identical, about 85%identical, about 90% identical, about 91% identical, about 92%identical, about 93% identical, about 94% identical, about 95%identical, about 96% identical, about 97% identical, about 98%identical, about 99% identical, or 100% identical to a sequence selectedfrom the group consisting of SEQ ID NOS:1-35. In some embodiments, suchan influenza antigen polypeptide retains immunogenic activity.

In some embodiments, an influenza antigen polypeptide can have an aminoacid sequence that comprises about 100 contiguous amino acids of asequence selected from the group consisting of SEQ ID NOS:1-35. In someembodiments, an influenza antigen polypeptide has an amino acid sequencewhich is about 60% identical, about 70% identical, about 80% identical,about 85% identical, about 90% identical, about 91% identical, about 92%identical, about 93% identical, about 94% identical, about 95%identical, about 96% identical, about 97% identical, about 98%identical, about 99% identical, or 100% identical to a contiguousstretch of about 100 amino acids of a sequence selected from the groupconsisting of SEQ ID NOS:1-35.

In some embodiments, an influenza antigen polypeptide can have an aminoacid sequence that comprises about 150, about 200, about 250, about 300,about 350, about 400, about 450, about 500, about 550, or morecontiguous amino acids of a sequence selected from the group consistingof SEQ ID NOS:1-35. In some embodiments, an influenza antigenpolypeptide has an amino acid sequence which is about 60% identical,about 70% identical, about 80% identical, about 85% identical, about 90%identical, about 91% identical, about 92% identical, about 93%identical, about 94% identical, about 95% identical, about 96%identical, about 97% identical, about 98% identical, about 99%identical, or 100% identical to a contiguous stretch of about 150, 200,250, 300, 350, or more amino acids of a sequence selected from the groupconsisting of SEQ ID NOS:1-35.

For example, sequences having sufficient identity to influenza antigenpolypeptide(s) which retain immunogenic characteristics are capable ofbinding with antibodies which react with one or more antigens providedherein. Immunogenic characteristics often include three dimensionalpresentation of relevant amino acids or side groups. One skilled in theart can readily identify sequences with modest differences in sequence(e.g., with difference in boundaries and/or some sequence alternatives,that, nonetheless preserve immunogenic characteristics).

In some embodiments, particular portions and/or domains of any of theexemplary sequences set forth in SEQ ID NOS:1-35 may be omitted from aninfluenza polypeptide. For example, HA polypeptides typically contain atransmembrane anchor sequence. HA polypeptides in which thetransmembrane anchor sequence has been omitted are contemplated herein.

As exemplary antigens, we have utilized sequences from hemagglutinin ofparticular subtypes as described in detail herein. Various subtypes ofinfluenza virus exist and continue to be identified as new subtypesemerge. It will be understood by one skilled in the art that the methodsand compositions provided herein may be adapted to utilize sequences ofadditional subtypes. Such variation is contemplated and encompassedwithin the methods and compositions provided herein.

Hemagglutinin Polypeptide Fusions with Thermostable Proteins

In certain aspects, provided are HA polypeptide(s) comprising fusionpolypeptides which comprise a HA polypeptide (or a portion or variantthereof) operably linked to a thermostable protein. Fusion polypeptidescan be produced in any available expression system known in the art. Incertain embodiments, fusion proteins are produced in a plant or portionthereof (e.g., plant, plant cell, root, or sprout).

Enzymes or other proteins that are not found naturally in human oranimal cells can be particularly useful in fusion polypeptides asprovided herein. For example, thermostable proteins that conferthermostability to a fusion product can be useful. Thermostability canallow a produced protein to maintain its conformation, and maintainproduced protein at room temperature. This feature can facilitate easy,time efficient and cost effective recovery of a fusion polypeptide. Arepresentative family of thermostable enzymes that can be used asdescribed herein is the glucanohydrolase family. These enzymesspecifically cleave 1,4-β glucosidic bonds that are adjacent to 1,3-βlinkages in mixed linked polysaccharides (Hahn et al., 1994 Proc. Natl.Acad. Sci., USA, 91:10417; incorporated herein by reference). Suchenzymes are found in cereals, such as oat and barley, and are also foundin a number of fungal and bacterial species, including C. thermocellum(Goldenkova et al., 2002, Mol. Biol. 36:698; incorporated herein byreference). Thus, suitable thermostable proteins for use in fusionpolypeptides as provided herein include glycosidase enzymes. Exemplarythermostable glycosidase proteins include those represented by GenBankaccession numbers selected from those set forth in Table 2, the contentsof each of which are incorporated herein by reference by entireincorporation of the GenBank accession information for each referencednumber. Exemplary thermostable enzymes for use in fusion proteinsinclude beta-glucanase enzymes from Clostridium thermocellum,Brevibacillus brevis, and Rhodthermus marinus. Representative fusionproteins can utilize modified thermostable enzymes isolated fromClostridium thermocellum, although any thermostable protein may besimilarly utilized. Exemplary thermostable glycosidase proteins arelisted in Table 2:

TABLE 2 Thermostable Glycosidase Proteins Accession StrainThermostable Protein Sequence X63355 Beta-5′MKNRVISLLMASLLLVLSVIVAPFYKAEAATVVNTPFVAV glucanaseFSNFDSSQWEKADWANGSVFNCVWKPSQVTFSNGKMILTL ClostridiumDREYGGSYPYKSGEYRTKSFFGYGYYEVRMKAAKNVGIVS thermo-SFFTYTGPSDNNPWDEIDIEFLGKDTTKVQFNWYKNGVGGN cellumEYLHNLGFDASQDFHTYGFEWRPDYIDFYVDGKKVYRGTRNIPVTPGKIMMNLWPGIGVDEWLGRYDGRTPLQAEYEYVKYYPNGVPQDNPTPTPTIAPSTPTNPNLPLKGDVNGDGHVNSSDYSLFKRYLLRVIDRFPVGDQSVADVNRDGRIDSTDLTMLK RYLIRAIPSL 3′ (SEQ ID NO: 36)P37073 Beta- 5′MVKSKYLVFISVFSLLFGVFVVGFSHQGVKAEEERPMGTA glucanaseFYESFDAFDDERWSKAGVWTNGQMFNATWYPEQVTADGL Brevi-MRLTIAKKTTSARNYKAGELRTNDFYHYGLFEVSMKPAKV bacillus EGTVSSFFTYTGEWDWDGDPWDEIDIEFLGKDTTRIQFNYFT brevisNGVGGNEFYYDLGFDASESFNTYAFEWREDSITWYVNGEAVHTATENIPQTPQKIMMNLWPGVGVDGWTGVFDGDNTPVY SYYDWVRYTPLQNYQIHQ 3′(SEQ ID NO: 37) P17989 Beta- 5′MNIKKTAVKSALAVAAAAAALTTNVSAKDFSGAELYTLEglucanase EVQYGKFEARMKMAAASGTVSSMFLYQNGSEIADGRPWVE FibrobacterVDIEVLGKNPGSFQSNIITGKAGAQKTSEKHHAVSPAADQAF succino-HTYGLEWTPNYVRWTVDGQEVRKTEGGQVSNLTGTQGLR genesFNLWSSESAAWVGQFDESKLPLFQFINWVKVYKYTPGQGEGGSDFTLDWTDNFDTFDGSRWGKGDWTFDGNRVDLTDKNIYSRDGMLILALTRKGQESFNGQVPRDDEPAPQSSSSAPASSSSVPASSSSVPASSSSAFVPPSSSSATNAIHGMRTTPAVAKEHR NLVNAKGAKVNPNGHKRYRVNFEH 3′(SEQ ID NO: 38) P07883 Extracellu-5′MVNRRDLIKWSAVALGAGAGLAGPAPAAHAADLEWEQY lar agarasePVPAAPGGNRSWQLLPSHSDDFNYTGKPQTFRGRWLDQHK Strepto-DGWSGPANSLYSARHSWVADGNLIVEGRRAPDGRVYCGY mycesVTSRTPVEYPLYTEVLMRVSGLKLSSNFWLLSRDDVNEIDVI coelicolorECYGNESLHGKHMNTAYHIFQRNPFTELARSQKGYFADGSYGYNGETGQVFGDGAGQPLLRNGFHRYGVHWISATEFDFYFNGRLVRRLNRSNDLRDPRSRFFDQPMHLILNTESHQWRVDRGIEPTDAELADPSINNIYYRWVRTYQAV 3′ (SEQ ID NO: 39) P23903 Glucan5′MKPSHFTEKRFMKKVLGLFLVVVMLASVGVLPTSKVQAA endo-13-GTTVTSMEYFSPADGPVISKSGVGKASYGFVMPKFNGGSAT beta-WNDVYSDVGVNVKVGNNWVDIDQAGGYIYNQNWGHWSD glucosidaseGGFNGYWFTLSATTEIQLYSKANGVKLEYQLVFQNINKTTIT A1 BacillusAMNPTQGPQITASFTGGAGFTYPTFNNDSAVTYEAVADDLK circulansVYVKPVNSSSWIDIDNNAASGWIYDHNFGQFTDGGGGYWFNVTESINVKLESKTSSANLVYTITFNEPTRNSYVITPYEGTTFTADANGSIGIPLPKIDGGAPIAKELGNFVYQININGQWVDLSNSSQSKFAYSANGYNNMSDANQWGYWADYIYGLWFQPIQENMQIRIGYPLNGQAGGNIGNNFVNYTFIGNPNAPRPDVSDQEDISIGTPTDPAIAGMNLIWQDEFNGTTLDTSKWNYETGYYLNNDPATWGWGNAELQHYTNSTQNVYVQDGKLNIKAMNDSKSFPQDPNRYAQYSSGKINTKDKLSLKYGRVDFRAKLPTGDGVWPALWMLPKDSVYGTWAASGEIDVMEARGRLPGSVSGTIHFGGQWPVNQSSGGDYHFPEGQTFANDYHVYSVVWEEDNIKWYVDGKFFYKVTNQQWYSTAAPNNPNAPFDEPFYLIMNLAVGGNFDGGRTPNASDIPATMQVDYVRVYKEQ 3′ (SEQ ID NO: 40) P27051 Beta-5′MSYRVKRMLMLLVTGLFLSLSTFAASASAQTGGSFYEPFN glucanaseNYNTGLWQKADGYSNGNMFNCTWRANNVSMTSLGEMRL BacillusSLTSPSYNKFDCGENRSVQTYGYGLYEVNMKPAKNVGIVSS licheni-FFTYTGPTDGTPWDEIDIEFLGKDTTKVQFNYYTNGVGNHE formisKIVNLGFDAANSYHTYAFDWQPNSIKWYVDGQLKHTATTQIPQTPGKIMMNLWNGAGVDEWLGSYNGVTPLSRSLHWVRY TKR 3′ (SEQ ID NO: 41) P45797Beta- 5′MMKKKSWFTLMITGVISLFFSVSAFAGNVFWEPLSYFNSS glucanaseTWQKADGYSNGQMFNCTWRANNVNFTNDGKLKLSLTSPA Paeni-NNKFDCGEYRSTNNYGYGLYEVSMKPAKNTGIVSSFFTYTG bacillusPSHGTQWDEIDIEFLGKDTTKVQFNYYTNGVGGHEKIINLGF polymyxaDASTSFHTYAFDWQPGYIKWYVDGVLKHTATTNIPSTPGKI BacillusMMNLWNGTGVDSWLGSYNGANPLYAEYDWVKYTSN 3′ polymyxa (SEQ ID NO: 42) P45798Beta- 5′MCTMPLMKLKKMMRRTAFLLSVLIGCSMLGSDRSDKAPH glucanaseWELVWSDEFDYSGLPDPEKWDYDVGGHGWGNQELQYYTR Rhodo-ARIENARVGGGVLIIEARHEPYEGREYTSARLVTRGKASWT thermusYGRFEIRARLPSGRGTWPAIWMLPDRQTYGSAYWPDNGEID marinusIMEHVGFNPDVVHGTVHTKAYNHLLGTQRGGSIRVPTARTDFHVYAIEWTPEEIRWFVDDSLYYRFPNERLTDPEADWRHWPFDQPFHLIMNIAVGGAWGGQQGVDPEAFPAQLVVDYVRVY RWVE 3′ (SEQ ID NO: 43) P38645Beta- 5′MTESAMTSRAGRGRGADLVAAVVQGHAAASDAAGDLSF glucosidasePDGFIWGAATAAYQIEGAWREDGRGLWDVFSHTPGKVASG Thermobis-HTGDIACDHYHRYADDVRLMAGLGDRVYRFSVAWPRIVPD poraGSGPVNPAGLDFYDRLVDELLGHGITPYPTLYHWDLPQTLE bisporaDRGGWAARDTAYRFAEYALAVHRRLGDRVRCWITLNEPWVAAFLATHRGAPGAADVPRFRAVHHLLLGHGLGLRLRSAGAGQLGLTLSLSPVIEARPGVRGGGRRVDALANRQFLDPALRGRYPEEVLKIMAGHARLGHPGRDLETIHQPVDLLGVNYYSHVRLAAEGEPANRLPGSEGIRFERPTAVTAWPGDRPDGLRTLLLRLSRDYPGVGLIITENGAAFDDRADGDRVHDPERIRYLTATLRAVHDAIMAGADLRGYFVWSVLDNFEWAYGYHKRGIVYVDYTTMRRIPRESALWYRDVVRRNGLRNGE 3′ (SEQ ID NO: 44) P40942 Celloxyla-5′MNKFLNKKWSLILTMGGIFLMATLSLIFATGKKAFNDQTS naseAEDIPSLAEAFRDYFPIGAAIEPGYTTGQIAELYKKHVNMLV ClostridiumAENAMKPASLQPTEGNFQWADADRIVQFAKENGMELRFHT sterco-LVWHNQTPTGFSLDKEGKPMVEETDPQKREENRKLLLQRL rariumENYIRAVVLRYKDDIKSWDVVNEVIEPNDPGGMRNSPWYQITGTEYIEVAFRATREAGGSDIKLYINDYNTDDPVKRDILYELVKNLLEKGVPIDGVGHQTHIDIYNPPVERIIESIKKFAGLGLDNIITELDMSIYSWNDRSDYGDSIPDYILTLQAKRYQELFDALKENKDIVSAVVFWGISDKYSWLNGFPVKRTNAPLLFDRNFM PKPAFWAIVDPSRLRE 3′(SEQ ID NO: 45) P14002 Beta- 5′MAVDIKKIIKQMTLEEKAGLCSGLDFWHTKPVERLGIPSIMglucosidase MTDGPHGLRKQREDAEIADINNSVPATCFPSAAGLACSWDR ClostridiumELVERVGAALGEECQAENVSILLGPGANIKRSPLCGRNFEYF thermo-SEDPYLSSELAASHIKGVQSQGVGACLKHFAANNQEHRRMT cellumVDTIVDERTLREIYFASFENAVKKARPWVVMCAYNKLNGEYCSENRYLLTEVLKNEWMHDGFVVSDWGAVNDRVSGLDAGLDLEMPTSHGITDKKIVEAVKSGKLSENILNRAVERILKVIFMALENKKENAQYDKDAHHRLARQAAAESMVLLKNEDDVLPLKKSGTIALIGAFVKKPRYQGSGSSHITPTRLDDIYEEIKKAGGDKVNLVYSEGYRLENDGIDEELINEAKKAASSSDVAVVFAGLPDEYESEGFDRTHMSIPENQNRLIEAVAEVQSNIVVVLLNGSPVEMPWIDKVKSVLEAYLGGQALGGALADVLFGEVNPSGKLAETFPVKLSHNPSYLNFPGEDDRVEYKEGLFVGYRYYDTKGIEPLFPFGHGLSYTKFEYSDISVDKKDVSDNSIINVSVKVKNVGKMAGKEIVQLYVKDVKSSVRRPEKELKGFEKVFLNPGEEKTVTFTLDKRAFAYYNTQIKDWHVESGEFLILIGRSSRDIVLKESVRVNSTVKIRKRFTVNSAVEDVMSDSSAAAVLGPVLKEITDALQIDMDNAHDMMAANIKNMPLRSLVGYSQGRL SEEMLEELVDKINNVE 3′(SEQ ID NO: 46) O33830 Alpha-5′MPSVKIGIIGAGSAVFSLRLVSDLCKTPGLSGSTVTLMDID glucosidaseEERLDAILTIAKKYVEEVGADLKFEKTMNLDDVIIDADFVIN ThermotogaTAMVGGHTYLEKVRQIGEKYGYYRGIDAQEFNMVSDYYTF maritimaSNYNQLKYFVDIARKIEKLSPKAWYLQAANPIFEGTTLVTRTVPIKAVGFCHGHYGVMEIVEKLGLEEEKVDWQVAGVNHGIWLNRFRYNGGNAYPLLDKWIEEKSKDWKPENPFNDQLSPAAIDMYRFYGVMPIGDTVRNSSWRYHRDLETKKKWYGEPWGGADSEIGWKWYQDTLGKVTEITKKVAKFIKENPSVRLSDLGSVLGKDLSEKQFVLEVEKILDPERKSGEQHIPFIDALLNDNKARFVVNIPNKGIIHGIDDDVVVEVPALVDKNGIHPEKIEPPLPDRVVKYYLRPRIMRMEMALEAFLTGDIRIIKELLYRDPRTKSDEQVEKVIEEILALPENEEMRKHYLKR 3′ (SEQ ID NO: 47) O43097 Xylanase5′MVGFTPVALAALAATGALAFPAGNATELEKRQTTPNSEG Thermo-WHDGYYYSWWSDGGAQATYTNLEGGTYEISWGDGGNLV mycesGGKGWNPGLNARAIHFEGVYQPNGNSYLAVYGWTRNPLV lanuginosusEYYIVENFGTYDPSSGATDLGTVECDGSIYRLGKTTRVNAPSIDGTQTFDQYWSVRQDKRTSGTVQTGCHFDAWARAGLNV NGDHYYQIVATEGYFSSGYARITVADVG 3′(SEQ ID NO: 48) P54583 Endo- 5′MPRALRRVPGSRVMLRVGVVVAVLALVAALANLAVPRPglucanase ARAAGGGYWHTSGREILDANNVPVRIAGINWFGFETCNYV E1 Acido-VHGLWSRDYRSMLDQIKSLGYNTIRLPYSDDILKPGTMPNSI thermusNFYQMNQDLQGLTSLQVMDKIVAYAGQIGLRIILDRHRPDC cellulo-SGQSALWYTSSVSEATWISDLQALAQRYKGNPTVVGFDLH lyticusNEPHDPACWGCGDPSIDWRLAAERAGNAVLSVNPNLLIFVEGVQSYNGDSYWWGGNLQGAGQYPVVLNVPNRLVYSAHDYATSVYPQTWFSDPTFPNNMPGIWNKNWGYLFNQNIAPVWLGEFGTTLQSTTDQTWLKTLVQYLRPTAQYGADSFQWTFWSWNPDSGDTGGILKDDWQTVDTVKDGYLAPIKSSIFDPVGASASPSSQPSPSVSPSPSPSPSASRTPTPTPTPTASPTPTLTPTATPTPTASPTPSPTAASGARCTASYQVNSDWGNGFTVTVAVTNSGSVATKTWTVSWTFGGNQTITNSWNAAVTQNGQSVTARNMSYNNVIQPGQNTTFGFQASYTGSNAAPTVACAAS 3′ (SEQ ID NO: 49) P14288 β-galacto-5′MLSFPKGFKFGWSQSGFQSEMGTPGSEDPNSDWHVWVH sidaseDRENIVSQVVSGDLPENGPGYWGNYKRFHDEAEKIGLNAV SulfolobusRINVEWSRIFPRPLPKPEMQTGTDKENSPVISVDLNESKLRE acidocal-MDNYANHEALSHYRQILEDLRNRGFHIVLNMYHWTLPIWL dariusHDPIRVRRGDFTGPTGWLNSRTVYEFARFSAYVAWKLDDLASEYATMNEPNVVWGAGYAFPRAGFPPNYLSFRLSEIAKWNIIQAHARAYDAIKSVSKKSVGIIYANTSYYPLRPQDNEAVEIAERLNRWSFFDSIIKGEITSEGQNVREDLRNRLDWIGVNYYTRTVVTKAESGYLTLPGYGDRCERNSLSLANLPTSDFGWEFFPEGLYDVLLKYWNRYGLPLYVMENGIADDADYQRPYYLVSHIYQVHRALNEGVDVRGYLHWSLADNYEWSSGFSMRFGLLKVDYLTKRLYWRPSALVYREITRSNGIPEELEHLNRVPPIKP LRH 3′ (SEQ ID NO: 50) O52629β-galac- 5′MFPEKFLWGVAQSGFQFEMGDKLRRNIDTNTDWWHWVR tosidaseDKTNIEKGLVSGDLPEEGINNYELYEKDHEIARKLGLNAYRI PyrococcusGIEWSRIFPWPTTFIDVDYSYNESYNLIEDVKITKDTLEELDEI woeseiANKREVAYYRSVINSLRSKGFKVIVNLNHFTLPYWLHDPIEARERALTNKRNGWVNPRTVIEFAKYAAYIAYKFGDIVDMWSTFNEPMVVVELGYLAPYSGFPPGVLNPEAAKLAILHMINAHALAYRQIKKFDTEKADKDSKEPAEVGIIYNNIGVAYPKDPNDSKDVKAAENDNFFHSGLFFEAIHKGKLNIEFDGETFIDAPYLKGNDWIGVNYYTREVVTYQEPMFPSIPLITFKGVQGYGYACRPGTLSKDDRPVSDIGWELYPEGMYDSIVEAHKYGVPVYVTENGIADSKDILRPYYIASHIKMTEKAFEDGYEVKGYFHWALTDNFEWALGFRMRFGLYEVNLITKERIPREKSVSIFREIVAN NGVTKKIEEELLRG 3′(SEQ ID NO: 51) P29094 Oligo-16-5′MERVWWKEAVVYQIYPRSFYDSNGDGIGDIRGIIAKLDYL glucosidaseKELGVDVVWLSPVYKSPNDDNGYDISDYRDIMDEFGTMAD GeobacillusWKTMLEEMHKRGIKLVMDLVVNHTSDEHPWFIESRKSKDN thermoglu-PYRDYYIWRPGKNGKEPNNWESVFSGSAWEYDEMTGEYYL cosidasiusHLFSKKQPDLNWENPKVRREVYEMMKFWLDKGVDGFRMDVINMISKVPELPDGEPQSGKKYASGSRYYMNGPRVHEFLQEMNREVLSKYDIMTVGETPGVTPKEGILYTDPSRRELNMVFQFEHMDLDSGPGGKWDIRPWSLADLKKTMTKWQKELEGKGWNSLYLNNHDQPRAVSRFGDDGKYRVESAKMLATFLHMMQGTPYIYQGEEIGMTNVRFPSIEDYRDIETLNMYKERVEEYGEDPQEVMEKIYYKGRDNARTPMQWDDSENAGFTAGTPWIPVNPNYKEINVKAALEDPNSVFHYYKKLIQLRKQHDIIVYGTYDLILEDDPYIYRYTRTLGNEQLIVITNFSEKTPVFRLPDHIIYKTKELLISNYDVDEAEELKEIRLRPWEARVYKIRLP 3′ (SEQ ID NO: 52) P49067 Alpha-5′MGDKINFIFGIHNHQPLGNFGWVFEEAYEKCYWPFLETLE amylaseEYPNMKVAIHTSGPLIEWLQDNRPEYIDLLRSLVKRGQVEIV PyrococcusVAGFYEPVLASIPKEDRIEQIRLMKEWAKSIGFDARGVWLTE furiosusRVWQPELVKTLKESGIDYVIVDDYHFMSAGLSKEELYWPYYTEDGGEVIAVFPIDEKLRYLIPFRPVDKVLEYLHSLIDGDESKVAVFHDDGEKFGIWPGTYEWVYEKGWLREFFDRISSDEKINLMLYTEYLEKYKPRGLVYLPIASYFEMSEWSLPAKQARLFVEFVNELKVKGIFEKYRVFVRGGIWKNFFYKYPESNYMHKRMLMVSKLVRNNPEARKYLLRAQCNDAYWHGLFGGVYLPHLRRAIWNNLIKANSYVSLGKVIRDIDYDGFEEVLIENDNFYAVFKPSYGGSLVEFSSKNRLVNYVDVLARRWEHYHGYVESQFDGVASIHELEKKIPDEIRKEVAYDKYRRFMLQDHVVPLGTTLEDFMFSRQQEIGEFPRVPYSYELLDGGIRLKREHLGIEVEKTVKLVNDGFEVEYIVNNKTGNPVLFAVELNVAVQSIMESPGVLRGKEIVVDDKYAVGKFALKFEDEMEVWKYPVKTLSQSESGWDLIQQGVSYIVPIRLEDKIRFKLKFEEASG 3′ (SEQ ID NO: 53) JC7532 Cellulase5′MMLRKKTKQLISSILILVLLLSLFPAALAAEGNTREDNFKH BacillusLLGNDNVKRPSEAGALQLQEVDGQMTLVDQHGEKIQLRGM speciesSTHGLQWFPEILNDNAYKALSNDWDSNMIRLAMYVGENGYATNPELIKQRVIDGIELAIENDMYVIVDWHVHAPGDPRDPVYAGAKDFFREIAALYPNNPHIIYELANEPSSNNNGGAGIPNNEEGWKAVKEYADPIVEMLRKSGNADDNIIIVGSPNWSQRPDLAADNPIDDHHTMYTVHFYTGSHAASTESYPSETPNSERGNVMSNTRYALENGVAVFATEWGTSQASGDGGPYFDEADVWIEFLNENNISWANWSLTNKNEVSGAFTPFELGKSNATNLDPGPDHVWAPEELSLSGEYVRARIKGVNYEPIDRTKYTKVLWDFNDGTKQGFGVNSDSPNKELIAVDNENNTLKVSGLDVSNDVSDGNFWANARLSANGWGKSVDILGAEKLTMDVIVDEPTTVAIAAIPQSSKSGWANPERAVRVNAEDFVQQTDGKYKAGLTITGEDAPNLKNIAFHEEDNNMNNIILFVGTDAADVIYLDNIKVIGTEVEIPVVHDPKGEAVLPSVFEDGTRQGWDWAGESGVKTALTIEEANGSNALSWEFGYPEVKPSDNWATAPRLDFWKSDLVRGENDYVAFDFYLDPVRATEGAMNINLVFQPPTNGYWVQAPKTYTINFDELEEANQVNGLYHYEVKINVRDITNIQDDTLLRNMMIIFADVESDFAGRVFVDNVRFEGAATTEPVEPEPVDPGEETPPVDEKEAKKEQKEAEKEEKEAVKEEKKEAKEEKKA VKNEAKKK 3′ (SEQ ID NO: 54)Q60037 Xylanase A 5′MQVRKRRGLLDVSTAVLVGILAGFLGVVLAASGVLSFGK ThermotogaEASSKGDSSLETVLALSFEGTTEGVVPFGKDVVLTASQDVA maritimaADGEYSLKVENRTSPWDGVEIDLTGKVKSGADYLLSFQVYQSSDAPQLFNVVARTEDEKGERYDVILDKVVVSDHWKEILVPFSPTFEGTPAKYSLIIVASKNTNFNFYLDKVQVLAPKESGPKVIYETSFENGVGDWQPRGDVNIEASSEVAHSGKSSLFISNRQKGWQGAQINLKGILKTGKTYAFEAWVYQNSGQDQTIIMTMQRKYSSDASTQYEWIKSATVPSGQWVQLSGTYTIPAGVTVEDLTLYFESQNPTLEFYVDDVKIVDTTSAEIKIEMEPEKEIPALKEVLKDYFKVGVALPSKVFLNPKDIELITKHFNSITAENEMKPESLLAGIENGKLKFRFETADKYIQFVEENGMVIRGHTLVWHNQTPDWFFKDENGNLLSKEAMTERLKEYIHTVVGHFKGKVYAWDVVNEAVDPNQPDGLRRSTWYQIMGPDYIELAFKFAREADPDAKLFYNDYNTFEPRKRDIIYNLVKDLKEKGLIDGIGMQCHISLATDIKQIEEAIKKFSTIPGIEIHITELDMSVYRDSSSNYPEAPRTALIEQAHKMMQLFEIFKKYSNVITNVTFWGLKDDYSWRATRRNDWPLIFDKDHQAKLAYWAIVAPEVLPPLPKESRISEGEAVVVGMMDDSYLMSKPIEILDEEGNVKATIRAVWKDSTIYIYGEVQDKTKKPAEDGVAIFINPNNERTPYLQPDDTYAVLWTNWKTEVNREDVQVKKFVGPGFRRYSFEMSITIPGVEFKKDSYIGFDAAVIDDGKWYSWSDTTNSQKTNTMNYGTLKLEGIMVATAKYGTPVIDGEIDEIWNTTEEIETKAVAMGSLDKNATAKVRVLWDENYLYVLAIVKDPVLNKDNSNPWEQDSVEIFIDENNHKTGYYEDDDAQFRVNYMNEQTFGTGGSPARFKTAVKLIEGGYIVEAAIKWKTIKPTPNTVIGFNIQVNDANEKGQRVGIISWSDPTNNSWRDPSKFGNLRLIK 3′ (SEQ ID NO: 55) P33558 Xylanase A5′MKRKVKKMAAMATSIIMAIMIILHSIPVLAGRITYDNETGT ClostridiumHGGYDYELWKDYGNTIMELNDGGTFSCQWSNIGNALFRKG sterco-RKFNSDKTYQELGDIVVEYGCDYNPNGNSYLCVYGWTRNP rariumLVEYYIVESWGSWRPPGATPKGTITQWMAGTYEIYETTRVNQPSIDGTATFQQYWSVRTSKRTSGTISVTEHFKQWERMGMRMGKMYEVALTVEGYQSSGYANVYKNEIRIGANPTPAPSQSPIRRDAFSIIEAEEYNSTNSSTLQVIGTPNNGRGIGYIENGNTVTYSNIDFGSGATGFSATVATEVNTSIQIRSDSPTGTLLGTLYVSSTGSWNTYQTVSTNISKITGVHDIVLVFSGPVNVDNFIFSRSSPVPAPGDNTRDAYSIIQAEDYDSSYGPNLQIFSLPGGGSAIGYIENGYSTTYKNIDFGDGATSVTARVATQNATTIQVRLGSPSGTLLGTIYVGSTGSFDTYRDVSATISNTAGVKDIVLVFSGPVN VDWFVFSKSGT 3′(SEQ ID NO: 56) P05117 Polygalact-5′MVIQRNSILLLIIIFASSISTCRSNVIDDNLFKQVYDNILEQEF uronase-2AHDFQAYLSYLSKNIESNNNIDKVDKNGIKVINVLSFGAKG precursorDGKTYDNIAFEQAWNEACSSRTPVQFVVPKNKNYLLKQITF SolanumSGPCRSSISVKIFGSLEASSKISDYKDRRLWIAFDSVQNLVVG lycopersic-GGGTINGNGQVWWPSSCKINKSLPCRDAPTALTFWNCKNL umKVNNLKSKNAQQIHIKFESCTNVVASNLMINASAKSPNTDGVHVSNTQYIQISDTIIGTGDDCISIVSGSQNVQATNITCGPGHGISIGSLGSGNSEAYVSNVTVNEAKIIGAENGVRIKTWQGGSGQASNIKFLNVEMQDVKYPIIIDQNYCDRVEPCIQQFSAVQVKNVVYENIKGTSATKVAIKFDCSTNFPCEGIIMENINLVGESGKPSEATCKNVHFNNAEHVTPHCTSLEISEDEALLYNY 3′ (SEQ ID NO: 57) P04954Cellulase D 5′MSRMTLKSSMKKRVLSLLIAVVFLSLTGVFPSGLIETKVSA ClostridiumAKITENYQFDSRIRLNSIGFIPNHSKKATIAANCSTFYVVKED thermo-GTIVYTGTATSMFDNDTKETVYIADFSSVNEEGTYYLAVPG cellumVGKSVNFKIAMNVYEDAFKTAMLGMYLLRCGTSVSATYNGIHYSHGPCHTNDAYLDYINGQHTKKDSTKGWHDAGDYNKYVVNAGITVGSMFLAWEHFKDQLEPVALEIPEKNNSIPDFLDELKYEIDWILTMQYPDGSGRVAHKVSTRNFGGFIMPENEHDERFFVPWSSAATADFVAMTAMAARIFRPYDPQYAEKCINAAKVSYEFLKNNPANVFANQSGFSTGEYATVSDADDRLWAAAEMWETLGDEEYLRDFENRAAQFSKKIEADFDWDNVANLGMFTYLLSERPGKNPALVQSIKDSLLSTADSIVRTSQNHGYGRTLGTTYYWGCNGTVVRQTMILQVANKISPNNDYVNAALDAISHVFGRNYYNRSYVTGLGINPPMNPHDRRSGADGIWEPWPGYLVGGGWPGPKDWVDIQDSYQTNEIAINWNAALIYALAGFVNYNSPQNEVLYGDVNDDGKVNSTDLTLLKRYVLKAVSTLPSSKAEKNADVNRDGRVNSSDVTILSRYLIRVIEKLPI 3′ (SEQ ID NO: 58) Q4J929 N-5′MLRSLVLNEKLRARVLERAEEFLLNNKADEEVWFRELVL glycosylaseCILTSNSSFISAYKSMNYILDKILYMDEKEISILLQESGYRFYN SulfolobusLKAKYLYRAKNLYGKVKKTIKEIADKDQMQAREFIATHIYG acidocal-IGYKEASHFLRNVGYLDLAIIDRHILRFINNLGIPIKLKSKREY dariusLLAESLLRSIANNLNVQVGLLDLFIFFKQTNTIVK 3′ (SEQ ID NO: 59) O33833 Beta-5′MFKPNYHFFPITGWMNDPNGLIFWKGKYHMFYQYNPRKP fructosidaseEWGNICWGHAVSDDLVHWRHLPVALYPDDETHGVFSGSA ThermotogaVEKDGKMFLVYTYYRDPTHNKGEKETQCVAMSENGLDFV maritimaKYDGNPVISKPPEEGTHAFRDPKVNRSNGEWRMVLGSGKDEKIGRVLLYTSDDLFHWKYEGVIFEDETTKEIECPDLVRIGEKDILIYSITSTNSVLFSMGELKEGKLNVEKRGLLDHGTDFYAAQTFFGTDRVVVIGWLQSWLRTGLYPTKREGWNGVMSLPRELYVENNELKVKPVDELLALRKRKVFETAKSGTFLLDVKENSYEIVCEFSGEIELRMGNESEEVVITKSRDELIVDTTRSGVSGGEVRKSTVEDEATNRIRAFLDSCSVEFFFNDSIAFSFRIHPEN VYNILSVKSNQVKLEVFELENIWL 3′(SEQ ID NO: 60) P49425 Endo-14-5′MAGPHRSRAAGPPPFAVDEHVALEMVAFRGEVFAGHGLL beta-manno-ADQRLIAHTGRPALNAQRITQQKQRDQCRGQRHRHHQGGR sidaseNLRKAHRTFHEHQSTQDQAHDAPHGQQAKTGHEGLGHEH Rhodo-AQAQHQQGQSNVVDRQDGEPVEAQHQKDGAQRAGNAPA thermusGRVELEQQPVEAQHQQQEGDVRIGKRRQNAFAPPALDHVH marinusGGPGRLQRHGLAVERHVPAVQQHQQRVQRGRQQIDHVLGHGLPGRQRLAFRDGPRRPVGVASPVLGQRPCPGHRIVQNLFRHGIDPCRVGRCRRSPSELHGMGCADVRARGHGRHMRGQRDEHPGRGRPCARRRHVDDDRDRTPQEKLYDVARGLDEPARRVHFDDEADRSVFRGLAQPAPDEPEGRRRDRLVLQRQSVNHRRGRLSRHRQQHQPQQQRPHGNQAFLGKYEKRRRKPTACLKSLRRFPDKDAPVLYFVNQLEKTKRRMTLLLVWLIFTGVAGEIRLEAEDGELLGVAVDSTLTGYSGRGYVTGFDAPEDSVRFSFEAPRGVYRVVFGVSFSSRFASYALRVDDWHQTGSLIKRGGGFFEASIGEIWLDEGAHTMAFQLMNGALDYVRLEPVSYGPPARPPAQLSDSQATASAQALFAFLLSEYGRHILAGQQQNPYRRDFDAINYVRNVTGKEPALVSFDLIDYSPTREAHGVVHYQTPEDWIAWAGRDGIVSLMWHWNAPTDLIEDPSQDCYWWYGFYTRCTTFDVAAALADTSSERYRLLLRDIDVIAAQLQKFQQADIPVLWRPLHEAAGGWFWWGAKGPEPFKQLWRLLYERLVHHHGLHNLIWVYTHEPGAAEWYPGDAYVDIVGRDVYADDPDALMRSDWNELQTLFGGRKLVALTETGTLPDVEVITDYGIWWSWFSIWTDPFLRDVDPDRLTRVYHSERVLTRDELPDWRSYVLHATTVQPAGDLALAVYPNPGAGRLHVEVGLPVAAPVVVEVFNLLGQRVFQYQAGMQPAGLWRRAFELALAPGV YLVQVRAGNLVARRRWVSVR 3′(SEQ ID NO: 61) P06279 Alpha- 5′MLTFHRIIRKGWMFLLAFLLTALLFCPTGQPAKAAAPFNGamylase TMMQYFEWYLPDDGTLWTKVANEANNLSSLGITALWLPPA GeobacillusYKGTSRSDVGYGVYDLYDLGEFNQKGAVRTKYGTKAQYL stearotherm-QAIQAAHAAGMQVYADVVFDHKGGADGTEWVDAVEVNP ophilusSDRNQEISGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYKFRGIGKAWDWEVDTENGNYDYLMYADLDMDHPEVVTELKSWGKWYVNTTNIDGFRLDAVKHIKFSFFPDWLSDVRSQTGKPLFTVGEYWSYDINKLHNYIMKTNGTMSLFDAPLHNKFYTASKSGGTFDMRTLMTNTLMKDQPTLAVTFVDNHDTEPGQALQSWVDPWFKPLAYAFILTRQEGYPCVFYGDYYGIPQYNIPSLKSKIDPLLIARRDYAYGTQHDYLDHSDIIGWTREGVTEKPGSGLAALITDGPGGSKWMYVGKQHAGKVFYDLTGNRSDTVTINSDGWGEFKVNGGSVSVWVPRKTTVSTIAWSITTRPWTDEFVRWTEPRLVAWP 3′ (SEQ ID NO: 62) P45702 Xylanase5′MPTNLFFNAHHSPVGAFASFTLGFPGKSGGLDLELARPPR P45703 GeobacillusQNVLIGVESLHESGLYHVLPFLETAEEDESKRYDIENPDPNP P40943 stearotherm-QKPNILIPFAKEEIQREFHVATDTWKAGDLTFTIYSPVKAVP ophilusNPETADEEELKLALVPAVIVEMTIDNTNGTRARRAFFGFEGTDPYTSMRRIDDTCPQLRGVGQGRILSIVSKDEGVRSALHFSMEDILTAQLEENWTFGLGKVGALIVDVPAGEKKTYQFAVCFYRGGYVTAGMDASYFYTRFFQNIEEVGLYALEQAEVLKEQSFRSNKLIEKEWLSDDQTFMMAHAIRSYYGNTQLLEHEGKPIWVVNEGEYRMMNTFDLTVDQLFFELKLNPWTVKNVLDLYVERYSYEDRVRFPGEETEYPSGISFTHDMGVANTFSRPHYSSYELYGISGCFSHMTHEQLVNWVLCAAVYIEQTKDWAWRDKRLAILEQCLESMVRRDHPDPEQRNGVMGLDSTRTMGGAEITTYDSLDVSLGQARNNLYLAGKCWAAYVALEKLFRDVGKEELAALAGEQAEKCAATIVSHVTDDGYIPAIMGEGNDSKIIPAIEGLVFPYFTNCHEALDENGRFGAYIQALRNHLQYVLREGICLFPDGGWKISSTSNNSWLSKIYLCQFIARHILGWEWDEQGKRADAAHVAWLTHPTLSIWSWSDQIIAGEITGSKYYPRGVTSIL WLEEGE 3′ (SEQ ID NO: 63)5′MCSSIPSLREVFANDFRIGAAVNPVTLEAQQSLLIRHVNSLTAENHMKFEHLQPEEGRFTFDIAIKSSTSPFSSHGVRGHTLVWHNQTPSWVFQDSQGHFVGRDVLLERMKSHISTVVQRYKGKVYCWDVINEAVADEGSEWLRSSTWRQIIGDDFIQQAFLYAHEADPEALLFYNDYNECFPEKREKIYTLVKSLRDKGIPIHGIGMQAHWSLNRPTLDEIRAAIERYASLGVILHITELDISMFEFDDHRKDLAAPTNEMVERQAERYEQIF SLFKEYRDVIQNVTFWGIADDHTWLDHFPVQGRKNWPLLFDEQHNPKPAFWRVVNI 3′ (SEQ ID NO: 64)5′MRNVVRKPLTIGLALTLLLPMGMTATSAKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHFNSIVAENVMKPISIQPEEGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLETHIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTEVEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAALGLDNQITELDVSMYGWPPRAYPTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAPYAKVEKGKGKDAPFVFGPDYKVKPAYWAIID HK 3′ (SEQ ID NO: 65) P09961Alpha- 5′MTKSIYFSLGIHNHQPVGNFDFVIERAYEMSYKPLINFFFK amylase 1HPDFPINVHFSGFLLLWLEKNHPEYFEKLKIMAERGQIEFVS Dictyo-GGFYEPILPIIPDKDKVQQIKKLNKYIYDKFGQTPKGMWLAE glomusRVWEPHLVKYIAEAGIEYVVVDDAHFFSVGLKEEDLFGYYL thereto-MEEQGYKLAVFPISMKLRYLIPFADPEETITYLDKFASEDKS philumKIALLFDDGEKFGLWPDTYRTVYEEGWLETFVSKIKENFLLVTPVNLYTYMQRVKPKGRIYLPTASYREMMEWVLFPEAQKELEELVEKLKTENLWDKFSPYVKGGFWRNFLAKYDESNHMQKKMLYVWKKVQDSPNEEVKEKAMEEVFQGQANDAYWHGIFGGLYLPHLRTAIYEHLIKAENYLENSEIRFNIFDFDCDGNDEIIVESPFFNLYLSPNHGGSVLEWDFKTKAFNLTNVLTRRKEAYHSKLSYVTSEAQGKSIHERWTAKEEGLENILFYDNHRRVSFTEKIFESEPVLEDLWKDSSRLEVDSFYENYDYEINKDENKIRVLFSGVFRGFELCKSYILYKDKSFVDVVYEIKNVSETPISLNFGWEINLNFLAPNHPDYYFLIGDQKYPLSSFGIEKVNNWKIFSGIGIELECVLDVEASLYRYPIETVSLSEEGFERVYQGAL IHFYKVDLPVGSTWRTTIRFWVK 3′(SEQ ID NO: 66) Q60042 Xylanase A5′MRKKRRGFLNASTAVLVGILAGFLGVVLAATGALGFAVR ThermotogaESLLLKQFLFLSFEGNTDGASPFGKDVVVTASQDVAADGEY neapolitanaSLKVENRTSVWDGVEIDLTGKVNTGTDYLLSFHVYQTSDSPQLFSVLARTEDEKGERYKILADKVVVPNYWKEILVPFSPTFEGTPAKFSLIITSPKKTDFVFYVDNVQVLTPKEAGPKVVYETSFEKGIGDWQPRGSDVKISISPKVAHSGKKSLFVSNRQKGWHGAQISLKGILKTGKTYAFEAWVYQESGQDQTIIMTMQRKYSSDSSTKYEWIKAATVPSGQWVQLSGTYTIPAGVTVEDLTLYFESQNPTLEFYVDDVKVVDTTSAEIKLEMNPEEEIPALKDVLKDYFRVGVALPSKVFINQKDIALISKHSNSSTAENEMKPDSLLAGIENGKLKFRFETADKYIEFAQQNGMVVRGHTLVWHNQTPEWFFKDENGNLLSKEEMTERLREYIHTVVGHFKGKVYAWDVVNEAVDPNQPDGLRRSTWYQIMGPDYIELAFKFAREADPNAKLFYNDYNTFEPKKRDIIYNLVKSLKEKGLIDGIGMQCHISLATDIRQIEEAIKKFSTIPGIEIHITELDISVYRDSTSNYSEAPRTALIEQAHKMAQLFKIFKKYSNVITNVTFWGLKDDYSWRATRRNDWPLIFDKDYQAKLAYWAIVAPEVLPPLPKESKISEGEAVVVGMMDDSYMMSKPIEIYDEEGNVKATIRAIWKDSTIYVYGEVQDATKKPAEDGVAIFINPNNERTPYLQPDDTYVVLWTNWKSEVNREDVEVKKFVGPGFRRYSFEMSITIPGVEFKKDSYIGFDVAVIDDGKWYSWSDTTNSQKTNTMNYGTLKLEGVMVATAKYGTPVIDGEIDDIWNTTEEIETKSVAMGSLEKNATAKVRVLWDEENLYVLAIVKDPVLNKDNSNPWEQDSVEIFIDENNHKTGYYEDDDAQFRVNYMNEQSFGTGASAARFKTAVKLIEGGYIVEAAIKWKTIKPSPNTVIGFNVQVNDANEKGQRV GIISWSDPTNNSWRDPSKFGNLRLIK 3′(SEQ ID NO: 67) AAN05438 Beta- 5′MDDHAEKFLWGVATSAYQIEGATQEDGRGPSIWDAFARRAAN05439 glycosidase PGAIRDGSTGEPACDHYRRYEEDIALMQSLGVRAYRFSVAW ThermusPRILPEGRGRINPKGLAFYDRLVDRLLASGITPFLTLYHWDLP thermo-LALEERGGWRSRETAFAFAEYAEAVARALADRVPFFATLNE philusPWCSAFLGHWTGEHAPGLRNLEAALRAAHHLLLGHGLAVEALRAAGARRVGIVLNFAPAYGEDPEAVDVADRYHNRYFLDPILGKGYPESPFRDPPPVPILSRDLELVARPLDFLGVNYYAPVRVAPGTGTLPVRYLPPEGPATAMGWEVYPEGLHHLLKRLGREVPWPLYVTENGAAYPDLWTGEAVVEDPERVAYLEAHVEAALRAREEGVDLRGYFVWSLMDNFEWAFGYTRRFGLYYV DFPSQRRIPKRSALWYRERIARAQT 3′(SEQ ID NO: 68) 5′MTENAEKFLWGVATSAYQIEGATQEDGRGPSIWDAFAQRPGAIRDGSTGEPACDHYRRYEEDIALMQSLGVRAYRFSVAWPRILPEGRGRINPKGLAFYDRLVDRLLASGITPFLTLYHWDLPLALEERGGWRSRETAFAFAEYAEAVARALADRVPFFATLNEPWCSAFLGHWTGEHAPGLRNLEAALRAAHHLLLGHGLAVEALRAAGARRVGIVLNFAPAYGEDPEAVDVADRYHNRFFLDPILGKGYPESPFRDPPPVPILSRDLELVARPLDFLGVNYYAPVRVAPGTGTLPVRYLPPEGPATAMGWEVYPEGLYHLLKRLGREVPWPLYVTENGAAYPDLWTGEAVVEDPERVAYLEAHVEAALRAREEGVDLRGYFVWSLMDNFEWAFGYTRRFGLYYV DFPSQRRIPKRSALWYRERIARAQT 3′(SEQ ID NO: 69) AAN05437 Sugar 5′MAQVGRGASPLSRARVPPLPHPLDGEHLPHDPAGGGHGKpermease ASSQDAPVGQLPGHLARPAFFHYLKNSFLVCSLTTVFALAV ThermusATFAGYALARFRFPGAELFGGSVLVTQVIPGILFLIPIYIMYIY thermo-VQNWVRSALGLEVRLVGSYGGLVFTYTAFFVPLSIWILRGF philusFASIPKELEEAAMVDGATPFQAFHRVILPLALPGLAATAVYIFLTAWDELLFAQVLTTEATATVPVGIRNFVGNYQNRYDLVMAAATVATLPVLVLFFFVQRQLIQGLTAGAVKG 3′ (SEQ ID NO: 70) AAN05440 Beta-5′MAENAEKFLWGVATSAYQIEGATQEDGRGPSIWDTFARR glycosidasePGAIRDGSTGEPACDHYHRYEEDIALMQSLGVGVYRFSVA ThermusWPRILPEGRGRINPKGLAFYDRLVDRLLAAGITPFLTLYHWD filiformisLPQALEDRGGWRSRETAFAFAEYAEAVARALADRVPFFATLNEPWCSAFLGHWTGEHAPGLRNLEAALRAAHHLLLGHGLAVEALRAAGAKRVGIVLNFAPVYGEDPEAVDVADRYHNRYFLDPILGRGYPESPFQDPPPTPNLSRDLELVARPLDFLGVNYYAPVRVAPGTGPLPVRYLPPEGPVTAMGWEVYPEGLYHLLKRLGREVPWPLYITENGAAYPDLWTGEAVVEDPERVAYLEAHVEAALRAREEGVDLRGYFVWSLMDNFEWAFGYTRRFGL YYVDFPSQRRIPKRSALWYRERIARAQL 3′(SEQ ID NO: 71) AAD43138 Beta-5′MKFPKDFMIGYSSSPFQFEAGIPGSEDPNSDWWVWVHDPE glycosidaseNTAAGLVSGDFPENGPGYWNLNQNDHDLAEKLGVNTIRVG Thermo-VEWSRIFPKPTFNVKVPVERDENGSIVHVDVDDKAVERLDE sphaeraLANKEAVNHYVEMYKDWVERGRKLILNLYHWPLPLWLHN aggregansPIMVRRMGPDRAPSGWLNEESVVEFAKYAAYIAWKMGELPVMWSTMNEPNVVYEQGYMFVKGGFPPGYLSLEAADKARRNMIQAHARAYDNIKRFSKKPVGLIYAFQWFELLEGPAEVFDKFKSSKLYYFTDIVSKGSSIINVEYRRDLANRLDWLGVNYYSRLVYKIVDDKPIILHGYGFLCTPGGISPAENPCSDFGWEVYPEGLYLLLKELYNRYGVDLIVTENGVSDSRDALRPAYLVSHVYSVWKAANEGIPVKGYLHWSLTDNYEWAQGFRQKFGLVMVDFKTKKRYLRPSALVFREIATHNGIPDELQHLTLIQ 3′ (SEQ ID NO: 72)

While sequences of exemplary thermostable polypeptides are providedherein, it will be appreciated that any sequence exhibitingthermostability may be employed. In some embodiments, a thermostablepolypeptide can have an amino acid sequence with about 60%, about 70%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOS:36-72. In some embodiments, such a thermostablepolypeptide can retain thermostability.

In some embodiments, a thermostable polypeptide can have an amino acidsequence that comprises about 100 contiguous amino acids of a sequenceselected from the group consisting of SEQ ID NOS:36-72. In someembodiments, a thermostable polypeptide can have an amino acid sequencewith about 60%, about 70%, about 80%, about 85%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or 100% sequence identity to a contiguous stretch ofabout 100 amino acids from a sequence selected from the group consistingof SEQ ID NOS:36-72.

In some embodiments, a thermostable polypeptide can have an amino acidsequence comprising about 150, about 200, about 250, about 300, about350, about 400, about 450, about 500, about 550, about 600, about 650,about 700, or more than 700 contiguous amino acids of a sequenceselected from the group consisting of SEQ ID NOS:36-72. In someembodiments, a thermostable polypeptide can have an amino acid sequencewith about 60%, about 70%, about 80%, about 85%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or 100% sequence identity to a contiguous stretch ofabout 150, 200, 250, 300, 350, or more than 350 amino acids from asequence selected from the group consisting of SEQ ID NO:36-72.

When designing fusion proteins and polypeptides, it typically isdesirable to preserve immunogenicity of the antigen. Still further, itis desirable in certain aspects to provide constructs which providethermostability of a fusion protein. This feature facilitates easy, timeefficient and cost effective recovery of a target antigen. In certainaspects, antigen fusion partners may be selected which provideadditional advantages, including enhancement of immunogenicity,potential to incorporate multiple vaccine determinants, yet lack priorimmunogenic exposure to vaccination subjects. Further beneficialqualities of fusion peptides of interest include proteins which provideease of manipulation for incorporation of one or more antigens, as wellas proteins which have potential to confer ease of production,purification, and/or formulation for vaccine preparations. One ofordinary skill in the art will appreciate that three dimensionalpresentation can affect each of these beneficial characteristics.Preservation of immunity or preferential qualities therefore may affect,for example, choice of fusion partner and/or choice of fusion location(e.g., N-terminus, C-terminus, internal, combinations thereof).Alternatively or additionally, preferences may affect length of segmentselected for fusion, whether it be length of antigen or length of fusionpartner selected.

As described herein, a variety of antigens can be fused with athermostable protein. For example, the thermostable carrier moleculeLicB, also referred to as lichenase, can be used for production offusion proteins. LicB is 1,3-1,4-β glucanase (LicB) from Clostridiumthermocellum, and has the following amino acid sequence (also set forthin EMBL accession: X63355 [gi:40697]):

(SEQ ID NO: 36) MKNRVISLLMASLLLVLSVIVAPFYKAEAATVVNTPFVAVFSNFDSSQWEKADWANGSVFNCVWKPSQVTFSNGKMILTLDREYGGSYPYKSGEYRTKSFFGYGYYEVRMKAAKNVGIVSSFFTYTGPSDNNPWDEIDIEFLGKDTTKVQFNWYKNGVGGNEYLHNLGFDASQDFHTYGFEWRPDYIDFYVDGKKVYRGTRNIPVTPGKIMMNLWPGIGVDEWLGRYDGRTPLQAEYEYVKYYPNGVPQDNPTPTPTIAPSTPTNPNLPLKGDVNGDGHVNSSDYSLFKRYLLRVIDRFPVGDQSVADVNRDGRIDSTDLTMLKRYLIRAI PSL.

LicB belongs to a family of globular proteins. Based on the threedimensional structure of LicB, its N- and C-termini are situated closeto each other on the surface, in close proximity to the active domain.LicB also has a loop structure exposed on the surface that is locatedfar from the active domain. We have generated constructs such that theloop structure and N- and C-termini of protein can be used as insertionsites for HA polypeptides. HA polypeptides can be expressed as N- orC-terminal fusions or as inserts into the surface loop. Importantly,LicB maintains its enzymatic activity at low pH and at high temperature(up to 75° C.). Thus, use of LicB as a carrier molecule contributesadvantages, including likely enhancement of target specificimmunogenicity, potential to incorporate multiple vaccine determinants,and straightforward formulation of vaccines that may be deliverednasally, orally or parenterally. Furthermore, production of LicB fusionsin plants should reduce the risk of contamination with animal or humanpathogens. See examples provided herein.

Fusion proteins comprising HA polypeptides can be produced in any of avariety of expression systems, including both in vitro and in vivosystems. One skilled in the art will readily appreciate thatoptimization of nucleic acid sequences for a particular expressionsystem is often desirable. For example, an exemplary optimized sequencefor expression of HA polypeptide-LicB fusions in plants is provided, andis shown in SEQ ID NO:73:

(SEQ ID NO: 73) 5′ MGFVLFSQLPSFLLVSTLLLFLVISHSCRAQN GGSYPYKSGEYRTKSFFGYGYYEVRMKAAKNVGIVSSFFTYTGPSDNNPWDEIDIEFLGKDTTKVQFNWYKNGVGGNEYLHNLGFDASQDFHTYGFEWRPDYIDFYVDGKKVYRGTRNIPVTPGKIMMNLWPGIGVDEWLGRYDGRTPLQAEYEYVKYYPNGrsk1VVNTPFVAVFSNFDSSQWEKADWANGSVFNCVWKP SQVTFSNGKMILTLDREYvdHHHHHHKDEL  3′.In SEQ ID NO:73, the bold/underlined portion corresponds to the signalsequence, the italicized/underlined portion corresponds to the 6× Histag and endoplasmic reticulum retention sequence, and the two portionsin lowercase letters correspond to restriction sites.

Thus, any relevant nucleic acid encoding a HA polypeptide(s), fusionprotein(s), or immunogenic portions thereof is intended to beencompassed within nucleic acid constructs provided herein.

For production in plant systems, transgenic plants expressing HApolypeptide(s) (e.g., HA polypeptide(s), fusion(s) thereof, and/orimmunogenic portion(s) thereof) may be utilized. Alternatively oradditionally, transgenic plants may be produced using methods well knownin the art to generate stable production crops. Additionally, plantsutilizing transient expression systems may be utilized for production ofHA polypeptide(s). When utilizing plant expression systems, whethertransgenic or transient expression in plants is utilized, any of nuclearexpression, chloroplast expression, mitochondrial expression, or viralexpression may be taken advantage of according to the applicability ofthe system to antigen desired. Furthermore, additional expressionsystems for production of antigens and fusion proteins can be utilized.For example, mammalian expression systems (e.g., mammalian cell linessuch as CHO cells), bacterial expression systems (e.g., E. coli), insectexpression systems (e.g., baculovirus), yeast expression systems, and invitro expression systems (e.g., reticulate lysates) may be used forexpression of antigens and fusion proteins.

Production of Influenza Antigens

Influenza antigens (including influenza protein(s), fragments, variants,and/or fusions thereof) can be produced in any suitable system;production is not limited to plant systems. Vector constructs andexpression systems are well known in the art and may be adapted toincorporate use of influenza antigens provided herein. For example,influenza antigens (including fragments, variants, and/or fusions) canbe produced in known expression systems, including mammalian cellsystems, transgenic animals, microbial expression systems, insect cellsystems, and plant systems, including transgenic and transient plantsystems. Particularly where influenza antigens are produced as fusionproteins, it may be desirable to produce such fusion proteins in plantsystems.

In some embodiments, influenza antigens are produced in plant systems.Plants are relatively easy to manipulate genetically, and have severaladvantages over alternative sources such as human fluids, animal celllines, recombinant microorganisms and transgenic animals. Plants havesophisticated post-translational modification machinery for proteinsthat is similar to that of mammals (although it should be noted thatthere are some differences in glycosylation patterns between plants andmammals). This enables production of bioactive reagents in planttissues. Also, plants can economically produce very large amounts ofbiomass without requiring sophisticated facilities. Moreover, plants arenot subject to contamination with animal pathogens. Like liposomes andmicrocapsules, plant cells are expected to provide protection forpassage of antigen to the gastrointestinal tract.

Plants may be utilized for production of heterologous proteins via useof various production systems. One such system includes use oftransgenic/genetically-modified plants where a gene encoding targetproduct is permanently incorporated into the genome of the plant.Transgenic systems may generate crop production systems. A variety offoreign proteins, including many of mammalian origin and many vaccinecandidate antigens, have been expressed in transgenic plants and shownto have functional activity (Tacket et al., 2000, J. Infect. Dis.,182:302; and Thanavala et al., 2005, Proc. Natl. Acad. Sci., USA,102:3378). Additionally, administration of unprocessed transgenic plantsexpressing hepatitis B major surface antigen to non-immunized humanvolunteers resulted in production of immune response (Kapusta et al.,1999, FASEB J., 13:1796).

Another system for expressing polypeptides in plants utilizes plantviral vectors engineered to express foreign sequences (e.g., transientexpression). This approach allows for use of healthy non-transgenicplants as rapid production systems. Thus, genetically engineered plantsand plants infected with recombinant plant viruses can serve as “greenfactories” to rapidly generate and produce specific proteins ofinterest. Plant viruses have certain advantages that make themattractive as expression vectors for foreign protein production. Severalmembers of plant RNA viruses have been well characterized, andinfectious cDNA clones are available to facilitate genetic manipulation.Once infectious viral genetic material enters a susceptible host cell,it replicates to high levels and spreads rapidly throughout the entireplant. There are several approaches to producing target polypeptidesusing plant viral expression vectors, including incorporation of targetpolypeptides into viral genomes. One approach involves engineering coatproteins of viruses that infect bacteria, animals or plants to functionas carrier molecules for antigenic peptides. Such carrier proteins havethe potential to assemble and form recombinant virus-like particlesdisplaying desired antigenic epitopes on their surface. This approachallows for time-efficient production of antigen and/or antibodycandidates, since the particulate nature of an antigen and/or antibodycandidate facilitates easy and cost-effective recovery from planttissue. Additional advantages include enhanced target-specificimmunogenicity, the potential to incorporate multiple antigendeterminants and/or antibody sequences, and ease of formulation intoantigen and/or antibody that can be delivered nasally, orally orparenterally. As an example, spinach leaves containing recombinant plantviral particles carrying epitopes of virus fused to coat protein havegenerated immune response upon administration (Modelska et al., 1998,Proc. Natl. Acad. Sci., USA, 95:2481; and Yusibov et al., 2002, Vaccine,19/20:3155).

Production of Hemagglutinin Antigens

HA antigens (including HA polypeptide(s), fusions thereof, and/orimmunogenic portions thereof) may be produced in any suitable system;production is not limited to plant systems. Vector constructs andexpression systems are well known in the art and may be adapted toincorporate use of HA polypeptides provided herein. For example, HApolypeptides can be produced in known expression systems, includingmammalian cell systems, transgenic animals, microbial expressionsystems, insect cell systems, and plant systems, including transgenicand transient plant systems. Particularly where HA polypeptides areproduced as fusion proteins, it may be desirable to produce such fusionproteins in plant systems.

In some embodiments, HA polypeptides are desirably produced in plantsystems. Plants are relatively easy to manipulate genetically, and haveseveral advantages over alternative sources such as human fluids, animalcell lines, recombinant microorganisms and transgenic animals. Plantshave sophisticated post-translational modification machinery forproteins that is similar to that of mammals (although it should be notedthat there are some differences in glycosylation patterns between plantsand mammals). This enables production of bioactive reagents in planttissues. Also, plants can economically produce very large amounts ofbiomass without requiring sophisticated facilities. Moreover, plants arenot subject to contamination with animal pathogens. Like liposomes andmicrocapsules, plant cells are expected to provide protection forpassage of antigen to the gastrointestinal tract.

Plants may be utilized for production of heterologous proteins via useof various production systems. One such system includes use oftransgenic/genetically-modified plants where a gene encoding targetproduct is permanently incorporated into the genome of the plant.Transgenic systems may generate crop production systems. A variety offoreign proteins, including many of mammalian origin and many vaccinecandidate antigens, have been expressed in transgenic plants and shownto have functional activity. (Tacket et al., 2000, J. Infect. Dis.,182:302; and Thanavala et al., 2005, Proc. Natl. Acad. Sci., USA,102:3378; both of which are incorporated herein by reference).Additionally, administration of unprocessed transgenic plants expressinghepatitis B major surface antigen to non-immunized human volunteersresulted in production of immune response (Kapusta et al., 1999, FASEBJ., 13:1796; incorporated herein by reference).

One system for expressing polypeptides in plants utilizes plant viralvectors engineered to express foreign sequences (e.g., transientexpression). This approach allows for use of healthy non-transgenicplants as rapid production systems. Thus, genetically engineered plantsand plants infected with recombinant plant viruses can serve as “greenfactories” to rapidly generate and produce specific proteins ofinterest. Plant viruses have certain advantages that make themattractive as expression vectors for foreign protein production. Severalmembers of plant RNA viruses have been well characterized, andinfectious cDNA clones are available to facilitate genetic manipulation.Once infectious viral genetic material enters a susceptible host cell,it replicates to high levels and spreads rapidly throughout the entireplant. There are several approaches to producing target polypeptidesusing plant viral expression vectors, including incorporation of targetpolypeptides into viral genomes. One approach involves engineering coatproteins of viruses that infect bacteria, animals or plants to functionas carrier molecules for antigenic peptides. Such carrier proteins havethe potential to assemble and form recombinant virus-like particlesdisplaying desired antigenic epitopes on their surface. This approachallows for time-efficient production of vaccine candidates, since theparticulate nature of a vaccine candidate facilitates easy andcost-effective recovery from plant tissue. Additional advantages includeenhanced target-specific immunogenicity, the potential to incorporatemultiple vaccine determinants, and ease of formulation into vaccinesthat can be delivered nasally, orally or parenterally. As an example,spinach leaves containing recombinant plant viral particles carryingepitopes of virus fused to coat protein have generated immune responseupon administration (Modelska et al., 1998, Proc. Natl. Acad. Sci., USA,95:2481; and Yusibov et al., 2002, Vaccine, 19/20:3155; both of whichare incorporated herein by reference).

Plant Expression Systems

The teachings herein are applicable to a wide variety of differentplants. In general, any plants that are amendable to expression ofintroduced constructs as described herein are useful in accordance withthe methods disclosed herein. In some embodiments, it is desirable touse young plants in order to improve the speed of protein/polypeptideproduction. As indicated here, in many embodiments, sprouted seedlingsare utilized. As is known in the art, most sprouts are quick growing,edible plants produced from storage seeds. However, those of ordinaryskill in the art will appreciate that the term “sprouted seedling” hasbeen used herein in a more general context, to refer to young plantswhether or not of a variety typically classified as “sprouts.” Any plantthat is grown long enough to have sufficient green biomass to allowintroduction and/or expression of an expression construct as providedfor herein (recognizing that the relevant time may vary depending on themode of delivery and/or expression of the expression construct) can beconsidered a “sprouted seedling” herein.

In many embodiments, edible plants are utilized (i.e., plants that areedible by—not toxic to—the subject to whom the protein or polypeptide isto be administered).

Any plant susceptible to incorporation and/or maintenance ofheterologous nucleic acid and capable of producing heterologous proteincan be utilized. In general, it may be desirable to utilize plants thatare amenable to growth under defined conditions, for example in agreenhouse and/or in aqueous systems. It may be desirable to selectplants that are not typically consumed by human beings or domesticatedanimals and/or are not typically part of the human food chain, so thatthey may be grown outside without concern that expressed polynucleotidemay be undesirably ingested. In some embodiments, however, it will bedesirable to employ edible plants. In particular embodiments, it will bedesirable to utilize plants that accumulate expressed polypeptides inedible portions of a plant.

Often, certain desirable plant characteristics will be determined by theparticular polynucleotide to be expressed. To give but a few examples,when a polynucleotide encodes a protein to be produced in high yield (aswill often be the case, for example, when antigen proteins are to beexpressed), it will often be desirable to select plants with relativelyhigh biomass (e.g., tobacco, which has additional advantages that it ishighly susceptible to viral infection, has a short growth period, and isnot in the human food chain). Where a polynucleotide encodes antigenprotein whose full activity requires (or is inhibited by) a particularpost-translational modification, the ability (or inability) of certainplant species to accomplish relevant modification (e.g., a particularglycosylation) may direct selection. For example, plants are capable ofaccomplishing certain post-translational modifications (e.g.,glycosylation), however, plants will not generate sialyation patternswhich are found in mammalian post-translational modification. Thus,plant production of antigen may result in production of a differententity than the identical protein sequence produced in alternativesystems.

In certain embodiments, crop plants, or crop-related plants areutilized. In certain specific embodiments, edible plants are utilized.

Plants for use in accordance with the methods provided herein include,for example, Angiosperms, Bryophytes (e.g., Hepaticae and Musci),Pteridophytes (e.g., ferns, horsetails, and lycopods), Gymnosperms(e.g., conifers, cycase, Ginko, and Gnetales), and Algae (e.g.,Chlorophyceae, Phaeophyceae, Rhodophyceae, Myxophyceae, Xanthophyceae,and Euglenophyceae). Exemplary plants include members of the familiesLeguminosae (Fabaceae; e.g., pea, alfalfa, and soybean); Gramineae(Poaceae; e.g., corn, wheat, and rice); Solanaceae, particularly of thegenus Lycopersicon (e.g., tomato), Solanum (e.g., potato and eggplant),Capsium (e.g., pepper), Nicotiana (e.g., tobacco); Umbelliferae,particularly of the genus Daucus (e.g., carrot), Apium (e.g., celery),or Rutaceae (e.g., oranges); Compositae, particularly of the genusLactuca (e.g., lettuce); and Brassicaceae (Cruciferae), particularly ofthe genus Brassica or Sinapis. In certain aspects, useful plants may bespecies of Brassica or Arabidopsis. Some exemplary Brassicaceae familymembers include Brassica campestris, B. carinata, B. juncea, B. napus,B. nigra, B. oleraceae, B. tournifortii, Sinapis alba, and Raphanussativus. Some suitable plants that are amendable to transformation andare edible as sprouted seedlings include alfalfa, mung bean, radish,wheat, mustard, spinach, carrot, beet, onion, garlic, celery, rhubarb, aleafy plant such as cabbage or lettuce, watercress or cress, herbs suchas parsley, mint, or clovers, cauliflower, broccoli, soybean, lentils,and edible flowers such as sunflower.

A wide variety of plant species may be suitable in the practicesdescribed herein. For example, a variety of different bean and otherspecies including, for example, adzuki bean, alfalfa, barley, broccoli,bill jump pea, buckwheat, cabbage, cauliflower, clover, collard greens,fenugreek, flax, garbanzo bean, green pea, Japanese spinach, kale,kamut, kohlrabi, marrowfat pea, mung bean, mustard greens, pinto bean,radish, red clover, soy bean, speckled pea, sunflower, turnip, yellowtrapper pea, and others may be amenable to the production ofheterologous proteins from viral vectors launched from an agrobacterialconstruct (e.g., introduced by agroinfiltration). In some embodiments,bill jump pea, green pea, marrowfat pea, speckled pea, and/or yellowtrapper pea are particularly useful. In certain embodiments, therefore,this document provides production of proteins or polypeptides (e.g.,antigens) in one or more of these plants using an agrobacterial vectorthat launches a viral construct (i.e., an RNA with characteristics of aplant virus) encoding the relevant protein or polypeptide of interest.In some embodiments, the RNA has characteristics of (and/or includessequences of) AlMV. In some embodiments, the RNA has characteristics of(and/or includes sequences of) TMV.

It will be appreciated that, in one aspect, this document provides youngplants (e.g., sprouted seedlings) that express a target protein orpolypeptide of interest. In some embodiments, the young plants weregrown from transgenic seeds; this document also provides seeds which canbe generated and/or utilized for the methods described herein. Seedstransgenic for any gene of interest can be sprouted and optionallyinduced for production of a protein or polypeptide of interest. Forexample, seeds capable of expressing any gene of interest can besprouted and induced through: i) virus infection, ii) agroinfiltration,or iii) bacteria that contain virus genome. Seeds capable of expressinga transgene for heavy or light chain of any monoclonal antibody can besprouted and induced for production of full-length molecule through: i)virus infection, ii) agroinfiltration, or iii) inoculation with bacteriathat contain virus genome. Seeds capable of expressing a transgene forone or more components of a complex molecule comprising multiplecomponents such as sIgA can be sprouted and used for producing a fullyfunctional molecule through: i) virus infection, ii) agroinfiltration,or iii) inoculation with bacteria that contain virus genome. Seeds fromhealthy non-transgenic plants can be sprouted and used for producingtarget sequences through: i) virus infection, ii) agroinfiltration, oriii) inoculation with bacteria that contain a virus genome.

In some embodiments, the young plants were grown from seeds that werenot transgenic. Typically, such young plants will harbor viral sequencesthat direct expression of the protein or polypeptide of interest. Insome embodiments, the plants may also harbor agrobacterial sequences,optionally including sequences that “launched” the viral sequences.

Introducing Vectors into Plants

In general, vectors may be delivered to plants according to knowntechniques. For example, vectors themselves may be directly applied toplants (e.g., via abrasive inoculations, mechanized spray inoculations,vacuum infiltration, particle bombardment, or electroporation).Alternatively or additionally, virions may be prepared (e.g., fromalready infected plants), and may be applied to other plants accordingto known techniques.

A wide variety of viruses are known that infect various plant species,and can be employed for polynucleotide expression (see, for example, inThe Classification and Nomenclature of Viruses, “Sixth Report of theInternational Committee on Taxonomy of Viruses” (Ed. Murphy et al.),Springer Verlag: New York, 1995; Grierson et al., Plant MolecularBiology, Blackie, London, pp. 126-146, 1984; Gluzman et al.,Communications in Molecular Biology: Viral Vectors, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., pp. 172-189, 1988; and Mathew,Plant Viruses Online; all of which are incorporated herein byreference). In certain embodiments, rather than delivering a singleviral vector to a plant cell, multiple different vectors are deliveredwhich, together, allow for replication (and, optionally cell-to-celland/or long distance movement) of viral vector(s). Some or all of theproteins may be encoded by the genome of transgenic plants. In certainaspects, described in further detail herein, these systems include oneor more viral vector components.

Vector systems that include components of two heterologous plant virusesin order to achieve a system that readily infects a wide range of planttypes and yet poses little or no risk of infectious spread. An exemplarysystem has been described previously (see, e.g., PCT Publication WO00/25574 and U.S. Patent Publication 2005/0026291, both of which areincorporated herein by reference). As noted herein, viral vectors can beapplied to plants (e.g., plants, portions of plant, or sprouts), throughinfiltration, mechanical inoculation, or spraying, for example. Whereinfection is to be accomplished by direct application of a viral genometo a plant, any available technique may be used to prepare the genome.For example, many viruses that are usefully employed in accordance withthe present disclosure have ssRNA genomes. ssRNA may be prepared bytranscription of a DNA copy of the genome, or by replication of an RNAcopy, either in vivo or in vitro. Given the readily availability ofeasy-to-use in vitro transcription systems (e.g., SP6, T7, andreticulocyte lysate), and also the convenience of maintaining a DNA copyof an RNA vector, ssRNA vectors may be prepared by in vitrotranscription, particularly with T7 or SP6 polymerase.

In certain embodiments, rather than introducing a single viral vectortype into a plant, multiple different viral vectors are introduced. Suchvectors may, for example, trans-complement each other with respect tofunctions such as replication, cell-to-cell movement, and/or longdistance movement. Vectors may contain different polynucleotidesencoding HA polypeptides as provided herein. Selection for plant(s) orportions thereof that express multiple polypeptides encoding one or moreHA polypeptide(s) may be performed as described above for singlepolynucleotides or polypeptides.

Plant Tissue Expression Systems

As discussed herein, HA polypeptides may be produced in any desirablesystem. Vector constructs and expression systems are well known in theart and may be adapted to incorporate use of HA polypeptides providedherein. For example, transgenic plant production is known and generationof constructs and plant production may be adapted according to knowntechniques in the art. In some embodiments, transient expression systemsin plants are desirable. Two of these systems include production ofclonal roots and clonal plant systems, and derivatives thereof, as wellas production of sprouted seedlings systems.

Clonal Plants

Clonal roots maintain RNA viral expression vectors and stably producetarget protein uniformly in an entire root over extended periods of timeand multiple subcultures. In contrast to plants, where a target gene iseliminated via recombination during cell-to-cell or long distancemovement, in root cultures the integrity of a viral vector is maintainedand levels of target protein produced over time are similar to thoseobserved during initial screening. Clonal roots allow for ease ofproduction of heterologous protein material for oral formulation ofantigen and vaccine compositions. Methods and reagents for generating avariety of clonal entities derived from plants which are useful forproduction of antigen (e.g., antigen proteins as provided herein) havebeen described previously and are known in the art (see, for example,PCT Publication WO 05/81905; incorporated herein by reference). Clonalentities include clonal root lines, clonal root cell lines, clonal plantcell lines, and clonal plants capable of production of antigen (e.g.,antigen proteins as described herein). This document further providesmethods and reagents for expression of antigen polynucleotide andpolypeptide products in clonal cell lines derived from various planttissues (e.g., roots, leaves), and in whole plants derived from singlecells (clonal plants). Such methods are typically based on use of plantviral vectors of various types.

For example, in one aspect, this document provides methods of obtaininga clonal root line that expresses a polynucleotide encoding a HApolypeptide, comprising the steps of: (i) introducing a viral vectorthat comprises a polynucleotide encoding an HA polypeptide as describedherein into a plant or portion thereof; and (ii) generating one or moreclonal root lines from a plant. Clonal root lines may be generated, forexample, by infecting a plant or plant portion (e.g., a harvested pieceof leaf) with an Agrobacterium (e.g., A. rhizogenes) that causesformation of hairy roots. Clonal root lines can be screened in variousways to identify lines that maintain virus, or lines that express apolynucleotide encoding a HA polypeptide at high levels, for example.This document further provides clonal root lines, e.g., clonal rootlines produced as described herein, and further encompasses methods ofexpressing polynucleotides and producing polypeptide(s) encoding HApolypeptide(s) using clonal root lines.

This document further provides methods of generating a clonal root cellline that expresses a polynucleotide encoding a HA polypeptide,comprising the steps of: (i) generating a clonal root line, cells ofwhich contain a viral vector whose genome comprises a polynucleotideencoding a HA polypeptide; (ii) releasing individual cells from a clonalroot line; and (iii) maintaining cells under conditions suitable forroot cell proliferation. Clonal root cell lines and methods ofexpressing polynucleotides and producing polypeptides using clonal rootcell lines also are provided herein.

In some aspects, this document provides methods of generating a clonalplant cell line that expresses a polynucleotide encoding a HApolypeptide, comprising the steps of: (i) generating a clonal root line,cells of which contain a viral vector whose genome comprises apolynucleotide encoding a HA polypeptide; (ii) releasing individualcells from a clonal root line; and (iii) maintaining cells in cultureunder conditions appropriate for plant cell proliferation. Also providedherein are methods for generating a clonal plant cell line thatexpresses a polynucleotide encoding a HA polypeptide, comprising thesteps of: (i) introducing into cells of a plant cell line maintained inculture a viral vector that comprises a polynucleotide encoding a HApolypeptide; and (ii) enriching for cells that contain viral vector.Enrichment may be performed, for example, by (i) removing a portion ofcells from the culture; (ii) diluting removed cells so as to reduce cellconcentration; (iii) allowing diluted cells to proliferate; and (iv)screening for cells that contain viral vector. Clonal plant cell linesmay be used for production of a HA polypeptide as provided herein.

This document provides a number of methods for generating clonal plants,cells of which contain a viral vector that comprises a polynucleotideencoding a HA polypeptide as disclosed herein. For example, thisdocument provides methods of generating a clonal plant that expresses apolynucleotide encoding a HA polypeptide, comprising the steps of: (i)generating a clonal root line, cells of which contain a viral vectorwhose genome comprises a polynucleotide encoding a HA polypeptide; (ii)releasing individual cells from a clonal root line; and (iii)maintaining released cells under conditions appropriate for formation ofa plant. This document further provides methods for generating a clonalplant that expresses a polynucleotide encoding a HA polypeptide,comprising the steps of: (i) generating a clonal plant cell line, cellsof which contain a viral vector whose genome comprises a polynucleotideencoding a HA polypeptide; and (ii) maintaining cells under conditionsappropriate for formation of a plant. In general, clonal plants asprovided herein can express any polynucleotide encoding a HA polypeptidein accordance with this document. Such clonal plants can be used forproduction of an antigen polypeptide.

As noted above, this document provides systems for expressing apolynucleotide or polynucleotide(s) encoding HA polypeptide(s) in clonalroot lines, clonal root cell lines, clonal plant cell lines (e.g., celllines derived from leaf or stem), and in clonal plants. A polynucleotideencoding a HA polypeptide can be introduced into an ancestral plant cellusing a plant viral vector whose genome includes polynucleotide encodingan HA polypeptide operably linked to (i.e., under control of) apromoter. A clonal root line or clonal plant cell line can establishedfrom a cell containing virus according to any of several techniques,including those that are further described below. The plant virus vectoror portions thereof can be introduced into a plant cell by infection, byinoculation with a viral transcript or infectious cDNA clone, byelectroporation, or by T-DNA mediated gene transfer, for example.

The following sections describe methods for generating clonal rootlines, clonal root cell lines, clonal plant cell lines, and clonalplants that express a polynucleotide encoding a HA polypeptide asprovided herein. A “root line” is distinguished from a “root cell line”in that a root line produces actual rootlike structures or roots while aroot cell line consists of root cells that do not form rootlikestructures. Use of the term “line” is intended to indicate that cells ofthe line can proliferate and pass genetic information on to progenycells. Cells of a cell line typically proliferate in culture withoutbeing part of an organized structure such as those found in an intactplant. Use of the term “root line” is intended to indicate that cells inthe root structure can proliferate without being part of a completeplant. It is noted that the term “plant cell” encompasses root cells.However, to distinguish methods for generating root lines and root celllines from those used to directly generate plant cell lines fromnon-root tissue (as opposed to generating clonal plant cell lines fromclonal root lines or clonal plants derived from clonal root lines), theterms “plant cell” and “plant cell line” as used herein generally referto cells and cell lines that consist of non-root plant tissue. Plantcells can be from, for example, leaf, stem, shoot, or flower part. It isnoted that seeds can be derived from clonal plants generated as derivedherein. Such seeds may contain viral vector as will plants obtained fromsuch seeds. Methods for obtaining seed stocks are well known in the art(see, for example, U.S. Patent Publication 2004/093643; incorporatedherein by reference).

Clonal Root Lines

This document provides systems for generating a clonal root line inwhich a plant viral vector is used to direct expression of apolynucleotide encoding a HA polypeptide. One or more viral expressionvector(s) including a polynucleotide encoding a HA polypeptide operablylinked to a promoter can be introduced into a plant or a portion thereofaccording to any of a variety of known methods. For example, plantleaves can be inoculated with viral transcripts. Vectors themselves maybe directly applied to plants (e.g., via abrasive inoculations,mechanized spray inoculations, vacuum infiltration, particlebombardment, or electroporation). Alternatively or additionally, virionsmay be prepared (e.g., from already infected plants), and may be appliedto other plants according to known techniques.

Where infection is to be accomplished by direct application of a viralgenome to a plant, any available technique may be used to prepare viralgenome. For example, many viruses that are usefully employed inaccordance with the present disclosure have ssRNA genomes. ssRNA may beprepared by transcription of a DNA copy of the genome, or by replicationof an RNA copy, either in vivo or in vitro. Given the readily available,easy-to-use in vitro transcription systems (e.g., SP6, T7, andreticulocyte lysate), and also the convenience of maintaining a DNA copyof an RNA vector, ssRNA vectors can be prepared by in vitrotranscription, particularly with T7 or SP6 polymerase. Infectious cDNAclones can be used. Agrobacterially mediated gene transfer can be usedto transfer viral nucleic acids such as viral vectors (either entireviral genomes or portions thereof) to plant cells using, e.g.,agroinfiltration, according to methods known in the art.

A plant or plant portion may then be then maintained (e.g., cultured orgrown) under conditions suitable for replication of viral transcript. Incertain embodiments, virus spreads beyond the initially inoculated cell,e.g., locally from cell to cell and/or systemically from an initiallyinoculated leaf into additional leaves. However, in some embodiments,virus does not spread. Thus viral vector may contain genes encodingfunctional MP and/or CP, but may be lacking one or both of such genes.In general, viral vector is introduced into (infects) multiple cells inthe plant or portion thereof.

Following introduction of viral vector into a plant, leaves areharvested. In general, leaves may be harvested at any time followingintroduction of a viral vector. However, it may be desirable to maintaina plant for a period of time following introduction of a viral vectorinto the plant, e.g., a period of time sufficient for viral replicationand, optionally, spread of virus from the cells into which it wasinitially introduced. A clonal root culture (or multiple cultures) isprepared, e.g., by known methods further described below.

In general, any available method may be used to prepare a clonal rootculture from a plant or plant tissue into which a viral vector has beenintroduced. One such method employs genes that exist in certainbacterial plasmids. These plasmids are found in various species ofAgrobacterium that infect and transfer DNA to a wide variety oforganisms. As a genus, Agrobacteria can transfer DNA to a large anddiverse set of plant types including numerous dicot and monocotangiosperm species and gymnosperms (see, for example, Gelvin, 2003,Microbiol. Mol. Biol. Rev., 67:16, and references therein, all of whichare incorporated herein by reference). The molecular basis of genetictransformation of plant cells is transfer from bacterium and integrationinto plant nuclear genome of a region of a large tumor-inducing (Ti) orrhizogenic (Ri) plasmid that resides within various Agrobacterialspecies. This region is referred to as the T-region when present in theplasmid and as T-DNA when excised from plasmid. Generally, asingle-stranded T-DNA molecule is transferred to a plant cell innaturally occurring Agrobacterial infection and is ultimatelyincorporated (in double-stranded form) into the genome. Systems based onTi plasmids are widely used for introduction of foreign genetic materialinto plants and for production of transgenic plants.

Infection of plants with various Agrobacterial species and transfer ofT-DNA has a number of effects. For example, A. tumefaciens causes crowngall disease while A. rhizogenes causes development of hairy roots atthe site of infection, a condition known as “hairy root disease.” Eachroot arises from a single genetically transformed cell. Thus root cellsin roots are clonal, and each root represents a clonal population ofcells. Roots produced by A. rhizogenes infection are characterized by ahigh growth rate and genetic stability (Giri et al., 2000, Biotech.Adv., 18:1, and references therein, all of which are incorporated hereinby reference). In addition, such roots are able to regenerategenetically stable plants (Giri 2000, supra).

In general, this document encompasses use of any strain of Agrobacteria,particularly any A. rhizogenes strain, that is capable of inducingformation of roots from plant cells. As mentioned above, a portion ofthe Ri plasmid (Ri T-DNA) is responsible for causing hairy root disease.While transfer of this portion of the Ri plasmid to plant cells canconveniently be accomplished by infection with Agrobacteria harboringthe Ri plasmid, this document encompasses use of alternative methods ofintroducing the relevant region into a plant cell. Such methods includeany available method of introducing genetic material into plant cellsincluding, but not limited to, biolistics, electroporation, PEG-mediatedDNA uptake, and Ti-based vectors. The relevant portions of Ri T-DNA canbe introduced into plant cells by use of a viral vector. Ri genes can beincluded in the same vector that contains a polynucleotide encoding a HApolypeptide or in a different viral vector, which can be the same or adifferent type to that of the vector that contains a polynucleotideencoding a HA polypeptide as provided herein. It is noted that theentire Ri T-DNA may not be required for production of hairy roots, andthis document encompasses use of portions of Ri T-DNA, provided thatsuch portions contain sufficient genetic material to induce rootformation, as known in the art. Additional genetic material, e.g., genespresent within the Ri plasmid but not within T-DNA, may be transferredto a plant cell, particularly genes whose expression products facilitateintegration of T-DNA into the plant cell DNA.

In order to prepare a clonal root line in accordance with certainembodiments, harvested leaf portions are contacted with A. rhizogenesunder conditions suitable for infection and transformation. Leafportions are maintained in culture to allow development of hairy roots.Each root is clonal, i.e., cells in the root are derived from a singleancestral cell into which Ri T-DNA was transferred. In some embodiments,a portion of such ancestral cells will contain a viral vector. Thuscells in a root derived from such an ancestral cell may contain viralvector since it will be replicated and will be transmitted during celldivision. Thus a high proportion (e.g., at least 50%, at least 75%, atleast 80%, at least 90%, at least 95%), all (100%), or substantially all(at least 98%) of cells will contain viral vector. It is noted thatsince viral vector is inherited by daughter cells within the clonalroot, movement of viral vector within the root is not necessary tomaintain viral vector throughout the root. Individual clonal hairy rootsmay be removed from the leaf portion and further cultured. Such rootsare also referred to herein as root lines. Isolated clonal rootscontinue to grow following isolation.

A variety of different clonal root lines have been generated usingmethods as described herein. These root lines were generated using viralvectors containing polynucleotide(s) encoding a HA polypeptide asprovided herein (e.g., encoding HA polypeptide(s), fusions thereof,and/or immunogenic portions thereof). Root lines were tested by Westernblot. Root lines displayed a variety of different expression levels ofvarious polypeptides. Root lines displaying high expression wereselected and further cultured. These root lines were subsequently testedagain and shown to maintain high levels of expression over extendedperiods of time, indicating stability. Expression levels were comparableto or greater than expression in intact plants infected with the sameviral vector used to generate clonal root lines. In addition, stabilityof expression of root lines was superior to that obtained in plantsinfected with the same viral vector. Up to 80% of such virus-infectedplants reverted to wild type after 2-3 passages. (Such passages involvedinoculating plants with transcripts, allowing infection (local orsystemic) to become established, taking a leaf sample, and inoculatingfresh plants that are subsequently tested for expression).

Root lines may be cultured on a large scale for production of antigenpolypeptides, as discussed further below. It is noted that clonal rootlines (and cell lines derived from clonal root lines) can generally bemaintained in medium that does not include various compounds, e.g.,plant growth hormones such as auxins and cytokinins, that typically areemployed in culture of root and plant cells. This feature greatlyreduces expense associated with tissue culture, and it may contributesignificantly to economic feasibility of protein production usingplants.

Any of a variety of methods may be used to select clonal roots thatexpress a polynucleotide encoding HA polypeptide(s) as provided herein.Western blots, ELISA assays, and other suitable techniques can be usedto detect an encoded polypeptide. In the case of detectable markers suchas GFP, alternative methods such as visual screens can be performed. Ifa viral vector that contains a polynucleotide that encodes a selectablemarker is used, an appropriate selection can be imposed (e.g., leafmaterial and/or roots derived therefrom can be cultured in the presenceof an appropriate antibiotic or nutritional condition and survivingroots identified and isolated). Certain viral vectors contain two ormore polynucleotide(s) encoding HA polypeptide(s), e.g., two or morepolynucleotides encoding different polypeptides. If one of these is aselectable or detectable marker, clonal roots that are selected ordetected by selecting for or detecting expression of the marker willhave a high probability of also expressing a second polynucleotide.Screening for root lines that contain particular polynucleotides canalso be performed using PCR and other nucleic acid detection methods.

Alternatively or additionally, clonal root lines can be screened forpresence of virus by inoculating host plants that will form locallesions as a result of virus infection (e.g., hypersensitive hostplants). For example, 5 mg of root tissue can be homogenized in 50 μl ofphosphate buffer and used to inoculate a single leaf of a tobacco plant.If virus is present in root cultures, within two to three dayscharacteristic lesions will appear on infected leaves. This means thatroot line contains recombinant virus that carries a polynucleotideencoding a HA polypeptide. If no local lesions are formed, there is novirus, and the root line is rejected as negative. This method is highlytime and cost efficient. After initially screening for the presence ofvirus, roots that contain virus may be subjected to secondary screening,e.g., by Western blot or ELISA to select high expressers. Additionalscreens can be applied, such as screens for rapid growth, growth inparticular media, or growth under particular environmental conditions,for example. These screening methods may, in general, be applied in thedevelopment of any of clonal root lines, clonal root cell lines, clonalplant cell lines, and/or clonal plants described herein.

As will be evident to one of ordinary skill in the art, a variety ofmodifications may be made to the methods for generating clonal rootlines that contain a viral vector, and such modifications are within thescope of this document. For example, while it is generally desirable tointroduce viral vector into an intact plant or portion thereof prior tointroduction of Ri T-DNA genes, in certain embodiments, the Ri-DNA isintroduced prior to introducing viral vector. In addition, it ispossible to contact intact plants with A. rhizogenes rather thanharvesting leaf portions and then exposing them to bacterium.

Other methods of generating clonal root lines from single cells of aplant or portion thereof that harbor a viral vector can be used (i.e.,methods not using A. rhizogenes or genetic material from the Riplasmid). For example, treatment with certain plant hormones orcombinations of plant hormones is known to result in generation of rootsfrom plant tissue.

Clonal Cell Lines Derived from Clonal Root Lines

As described above, this document provides methods for generating clonalroot lines, wherein cells in root lines contain a viral vector. As iswell known in the art, a variety of different cell lines can begenerated from roots. For example, root cell lines can be generated fromindividual root cells obtained from a root using a variety of knownmethods. Such root cell lines may be obtained from various differentroot cell types within the root. In general, root material is harvestedand dissociated (e.g., physically and/or enzymatically digested) torelease individual root cells, which are then further cultured. Completeprotoplast formation is generally not necessary. If desired, root cellscan be plated at very dilute cell concentrations, so as to obtain rootcell lines from single root cells. Root cell lines derived in thismanner are clonal root cell lines containing viral vector. Such rootcell lines therefore exhibit stable expression of a polynucleotideencoding a HA polypeptide as provided herein. Clonal plant cell linescan be obtained in a similar manner from clonal roots, e.g., byculturing dissociated root cells in the presence of appropriate planthormones. Screens and successive rounds of enrichment can be used toidentify cell lines that express a polynucleotide encoding a HApolypeptide at high levels. However, if the clonal root line from whichthe cell line is derived already expresses at high levels, suchadditional screens may be unnecessary.

As in the case of the clonal root lines, cells of a clonal root cellline are derived from a single ancestral cell that contains viral vectorand will, therefore, also contain viral vector since it will bereplicated and will be transmitted during cell division. Thus a highproportion (e.g. at least 50%, at least 75%, at least 80%, at least 90%,at least 95%), all (100%), or substantially all (at least 98%) of cellswill contain viral vector. It is noted that since viral vector isinherited by daughter cells within a clonal root cell line, movement ofviral vector among cells is not necessary to maintain viral vector.Clonal root cell lines can be used for production of a polynucleotideencoding a HA polypeptide, as described below.

Clonal Plant Cell Lines

This document provides methods for generating a clonal plant cell linein which a plant viral vector is used to direct expression of apolynucleotide encoding a HA polypeptide as provided herein. Accordingto these methods, one or more viral expression vector(s) including apolynucleotide encoding a HA polypeptide operably linked to a promoteris introduced into cells of a plant cell line that is maintained in cellculture. A number of plant cell lines from various plant types are knownin the art, any of which can be used. Newly derived cell lines can begenerated according to known methods for use in practicing the methodsdisclosed herein. A viral vector can be introduced into cells of a plantcell line according to any of a number of methods. For example,protoplasts can be made and viral transcripts then electroporated intocells. Other methods of introducing a plant viral vector into cells of aplant cell line also can be used.

A method for generating clonal plant cell lines and a viral vectorsuitable for introduction into plant cells (e.g., protoplasts) can beused as follows: Following introduction of viral vector, a plant cellline may be maintained in tissue culture. During this time viral vectormay replicate, and polynucleotide(s) encoding a HA polypeptide(s) asprovided herein may be expressed. Clonal plant cell lines are derivedfrom culture, e.g., by a process of successive enrichment. For example,samples may be removed from culture, optionally with dilution so thatthe concentration of cells is low, and plated in Petri dishes inindividual droplets. Droplets are then maintained to allow celldivision.

It will be appreciated that droplets may contain a variable number ofcells, depending on the initial density of the culture and the amount ofdilution. Cells can be diluted such that most droplets contain either 0or 1 cell if it is desired to obtain clonal cell lines expressing apolynucleotide encoding a HA polypeptide after only a single round ofenrichment. However, it can be more efficient to select a concentrationsuch that multiple cells are present in each droplet and then screendroplets to identify those that contain expressing cells. In general,any appropriate screening procedure can be employed. For example,selection or detection of a detectable marker such as GFP can be used.Western blots or ELISA assays can be used. Individual droplets (100 μl)contain more than enough cells for performance of these assays. Multiplerounds of enrichment are performed to isolate successively higherexpressing cell lines. Single clonal plant cell lines (i.e., populationsderived from a single ancestral cell) can be generated by furtherlimiting dilution using standard methods for single cell cloning.However, it is not necessary to isolate individual clonal lines. Apopulation containing multiple clonal cell lines can be used forexpression of a polynucleotide encoding one or more HA polypeptide(s).

In general, certain considerations described above for generation ofclonal root lines apply to the generation of clonal plant cell lines.For example, a diversity of viral vectors containing one or morepolynucleotide(s) encoding a HA polypeptide(s) as provided herein can beused as can combinations of multiple different vectors. Similarscreening methods can be used. As in the case of clonal root lines andclonal root cell lines, cells of a clonal plant cell line are derivedfrom a single ancestral cell that contains viral vector and will,therefore, also contain viral vector since it will be replicated andwill be transmitted during cell division. Thus a high proportion (e.g.at least 50%, at least 75%, at least 80%, at least 90%, at least 95%),all (100%), or substantially all (at least 98%) of cells will containviral vector. It is noted that since viral vector is inherited bydaughter cells within a clonal plant cell line, movement of viral vectoramong cells is not necessary to maintain viral vector. The clonal plantcell line can be used for production of a polypeptide encoding a HApolypeptide as described below.

Clonal Plants

Clonal plants can be generated from clonal roots, clonal root celllines, and/or clonal plant cell lines produced according to variousmethods described herein. Methods for generation of plants from roots,root cell lines, and plant cell lines such as clonal root lines, clonalroot cell lines, and clonal plant cell lines described herein are wellknown in the art (see, e.g., Peres et al., 2001, Plant Cell, Tissue,Organ Culture, 65:37; incorporated herein by reference; and standardreference works on plant molecular biology and biotechnology citedelsewhere herein). This document therefore provides a method ofgenerating a clonal plant comprising steps of (i) generating a clonalroot line, clonal root cell line, or clonal plant cell line according toany of the methods described herein; and (ii) generating a whole plantfrom a clonal root line, clonal root cell line, or clonal plant. Clonalplants may be propagated and grown according to standard methods.

As in the case of clonal root lines, clonal root cell lines, and clonalplant cell lines, cells of a clonal plant are derived from a singleancestral cell that contains viral vector and will, therefore, alsocontain viral vector since it will be replicated and will be transmittedduring cell division. Thus a high proportion (e.g. at least 50%, atleast 75%, at least 80%, at least 90%, at least 95%), all (100%), orsubstantially all (at least 98%) of cells will contain viral vector. Itis noted that since viral vector is inherited by daughter cells withinthe clonal plant, movement of viral vector is not necessary to maintainviral vector.

Sprouts and Sprouted Seedling Plant Expression Systems

Any of a variety of different systems can be used to express proteins orpolypeptides in young plants (e.g., sprouted seedlings). In someembodiments, transgenic cell lines or seeds are generated, which arethen sprouted and grown for a period of time so that a protein orpolypeptide included in the transgenic sequences is produced in youngplant tissues (e.g., in sprouted seedlings). Typical technologies forthe production of transgenic plant cells and/or seeds includeAgrobacterium tumefaciens mediated gene transfer and microprojectilebombardment or electroporation.

Systems and reagents for generating a variety of sprouts and sproutedseedlings which are useful for production of HA polypeptide(s) asdescribed herein have been described previously and are known in the art(see, for example, PCT Publication WO 04/43886; incorporated herein byreference). This document further provides sprouted seedlings, which maybe edible, as a biomass containing a HA polypeptide. In certain aspects,biomass is provided directly for consumption of antigen containingcompositions. In some aspects, biomass is processed prior toconsumption, for example, by homogenizing, crushing, drying, orextracting. In certain aspects, HA polypeptides are purified frombiomass and formulated into a pharmaceutical composition.

Additionally provided are methods for producing HA polypeptide(s) insprouted seedlings that can be consumed or harvested live (e.g.,sprouts, sprouted seedlings of the Brassica genus). In certain aspects,the methods can include growing a seed to an edible sprouted seedling ina contained, regulatable environment (e.g., indoors and/or in acontainer). A seed can be a genetically engineered seed that contains anexpression cassette encoding a HA polypeptide, which expression isdriven by an exogenously inducible promoter. A variety of exogenouslyinducible promoters can be used that are inducible, for example, bylight, heat, phytohormones, and/or nutrients.

In some embodiments, this document provides methods of producing HApolypeptide(s) in sprouted seedlings by first generating a seed stockfor a sprouted seedling by transforming plants with an expressioncassette that encodes HA polypeptide using an Agrobacteriumtransformation system, wherein expression of a HA polypeptide is drivenby an inducible promoter. Transgenic seeds can be obtained from atransformed plant, grown in a contained, regulatable environment, andinduced to express a HA polypeptide.

In some embodiments, methods are provided that include infectingsprouted seedlings with a viral expression cassette encoding a HApolypeptide, expression of which may be driven by any of a viralpromoter or an inducible promoter. Sprouted seedlings can be grown fortwo to fourteen days in a contained, regulatable environment or at leastuntil sufficient levels of HA polypeptide have been obtained forconsumption or harvesting.

This document further provides systems for producing HA polypeptide(s)in sprouted seedlings that include a housing unit with climate controland a sprouted seedling containing an expression cassette that encodesone or more HA polypeptides, wherein expression is driven by aconstitutive or inducible promoter. Systems can provide uniqueadvantages over the outdoor environment or greenhouse, which cannot becontrolled. Thus, this document enables a grower to precisely time theinduction of expression of HA polypeptide. It can greatly reduce timeand cost of producing HA polypeptide(s).

In certain aspects, transiently transfected sprouts contain viral vectorsequences encoding an HA polypeptide as provided herein. Seedlings canbe grown for a time period so as to allow for production of viralnucleic acid in sprouts, followed by a period of growth wherein multiplecopies of virus are produced, thereby resulting in production of HApolypeptide(s).

In certain aspects, genetically engineered seeds or embryos that containa nucleic acid encoding HA polypeptide(s) are grown to sprouted seedlingstage in a contained, regulatable environment. The contained,regulatable environment may be a housing unit or room in which seeds canbe grown indoors. All environmental factors of a contained, regulatableenvironment may be controlled. Since sprouts do not require light togrow, and lighting can be expensive, genetically engineered seeds orembryos may be grown to sprouted seedling stage indoors in the absenceof light.

Other environmental factors that can be regulated in a contained,regulatable environment include temperature, humidity, water, nutrients,gas (e.g., O₂ or CO₂ content or air circulation), chemicals (smallmolecules such as sugars and sugar derivatives, or hormones such asphytohormones, including gibberellins and abscisic acid), and the like.

According to certain methods provided herein, expression of a nucleicacid encoding a HA polypeptide may be controlled by an exogenouslyinducible promoter. Exogenously inducible promoters are caused toincrease or decrease expression of a nucleic acid in response to anexternal, rather than an internal stimulus. A number of environmentalfactors can act as inducers for expression of nucleic acids carried byexpression cassettes of genetically engineered sprouts. A promoter maybe a heat-inducible promoter, such as a heat-shock promoter. Forexample, using as heat-shock promoter, temperature of a containedenvironment may simply be raised to induce expression of a nucleic acid.Other promoters include light inducible promoters. Light-induciblepromoters can be maintained as constitutive promoters if light in acontained regulatable environment is always on. Alternatively oradditionally, expression of a nucleic acid can be turned on at aparticular time during development by simply turning on the light. Apromoter may be a chemically inducible promoter is used to induceexpression of a nucleic acid. According to these embodiments, a chemicalcould simply be misted or sprayed onto seed, embryo, or seedling toinduce expression of nucleic acid. Spraying and misting can be preciselycontrolled and directed onto target seed, embryo, or seedling to whichit is intended. The contained environment is devoid of wind or aircurrents, which could disperse chemical away from intended target, sothat the chemical stays on the target for which it was intended.

The time at which expression is induced can be selected to maximizeexpression of a HA polypeptide in sprouted seedling by the time ofharvest. Inducing expression in an embryo at a particular stage ofgrowth, for example, inducing expression in an embryo at a particularnumber of days after germination, may result in maximum synthesis of aHA polypeptide at the time of harvest. For example, inducing expressionfrom the promoter 4 days after germination may result in more proteinsynthesis than inducing expression from the promoter after 3 days orafter 5 days. Those skilled in the art will appreciate that maximizingexpression can be achieved by routine experimentation. In certainmethods, sprouted seedlings are harvested at about 1 day, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days,or 12 days after germination.

In cases where the expression vector has a constitutive promoter insteadof an inducible promoter, sprouted seedling may be harvested at acertain time after transformation of sprouted seedling. For example, ifa sprouted seedling were virally transformed at an early stage ofdevelopment, for example, at embryo stage, sprouted seedlings may beharvested at a time when expression is at its maximumpost-transformation, e.g., at about 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, or 14 days post-transformation. It could be that sprouts developone, two, three or more months post-transformation, depending ongermination of seed.

Generally, once expression of HA polypeptide(s) begins, seeds, embryos,or sprouted seedlings are allowed to grow until sufficient levels of HApolypeptide(s) are expressed. In certain aspects, sufficient levels arelevels that would provide a therapeutic benefit to a patient ifharvested biomass were eaten raw. Alternatively or additionally,sufficient levels are levels from which HA polypeptide can beconcentrated or purified from biomass and formulated into apharmaceutical composition that provides a therapeutic benefit to apatient upon administration. Typically, HA polypeptide is not a proteinexpressed in sprouted seedling in nature. At any rate, HA polypeptide istypically expressed at concentrations above that which would be presentin the sprouted seedling in nature.

Once expression of HA polypeptide is induced, growth is allowed tocontinue until sprouted seedling stage, at which time sprouted seedlingsare harvested. Sprouted seedlings can be harvested live. Harvesting livesprouted seedlings has several advantages including minimal effort andbreakage. Sprouted seedlings may be grown hydroponically, makingharvesting a simple matter of lifting a sprouted seedling from itshydroponic solution. No soil is required for growth of sproutedseedlings, but soil may be provided if deemed necessary or desirable bythe skilled artisan. Because sprouts can be grown without soil, nocleansing of sprouted seedling material is required at the time ofharvest. Being able to harvest the sprouted seedling directly from itshydroponic environment without washing or scrubbing minimizes breakageof harvested material. Breakage and wilting of plants induces apoptosis.During apoptosis, certain proteolytic enzymes become active, which candegrade pharmaceutical protein expressed in the sprouted seedling,resulting in decreased therapeutic activity of the protein.Apoptosis-induced proteolysis can significantly decrease yield ofprotein from mature plants. Using methods provided herein, apoptosis maybe avoided when no harvesting takes place until the moment proteins areextracted from the plant.

For example, live sprouts may be ground, crushed, or blended to producea slurry of sprouted seedling biomass, in a buffer containing proteaseinhibitors. Buffer may be maintained at about 4° C. In some aspects,sprouted seedling biomass is air-dried, spray dried, frozen, orfreeze-dried. As in mature plants, some of these methods, such asair-drying, may result in a loss of activity of pharmaceutical protein.However, because sprouted seedlings are very small and have a largesurface area to volume ratio, this is much less likely to occur. Thoseskilled in the art will appreciate that many techniques for harvestingbiomass that minimize proteolysis of expressed protein are available andcould be applied to the subject matter described herein.

In some embodiments, sprouted seedlings are edible. In certainembodiments, sprouted seedlings expressing sufficient levels of HApolypeptides are consumed upon harvesting (e.g., immediately afterharvest, within minimal period following harvest) so that absolutely noprocessing occurs before sprouted seedlings are consumed. In this way,any harvest-induced proteolytic breakdown of HA polypeptide beforeadministration of HA polypeptide to a patient in need of treatment isminimized. For example, sprouted seedlings that are ready to be consumedcan be delivered directly to a patient. Alternatively or additionally,genetically engineered seeds or embryos are delivered to a patient inneed of treatment and grown to sprouted seedling stage by a patient. Inone aspect, a supply of genetically engineered sprouted seedlings isprovided to a patient, or to a doctor who will be treating patients, sothat a continual stock of sprouted seedlings expressing certaindesirable HA polypeptide may be cultivated. This may be particularlyvaluable for populations in developing countries, where expensivepharmaceuticals are not affordable or deliverable. The ease with whichsprouted seedlings can be grown can make them particularly desirable forsuch developing populations.

The regulatable nature of the contained environment can impartadvantages over growing plants in the outdoor environment. In general,growing genetically engineered sprouted seedlings that expresspharmaceutical proteins in plants provides a pharmaceutical productfaster (because plants are harvested younger) and with less effort,risk, and regulatory considerations than growing genetically engineeredplants. A contained, regulatable environment reduces or eliminates riskof cross-pollinating plants in nature.

For example, a heat inducible promoter likely would not be used outdoorsbecause outdoor temperature cannot be controlled. The promoter would beturned on any time the outdoor temperature rose above a certain level.Similarly, the promoter would be turned off every time the outdoortemperature dropped. Such temperature shifts could occur in a singleday, for example, turning expression on in the daytime and off at night.A heat inducible promoter, such as those described herein, would noteven be practical for use in a greenhouse, which is susceptible toclimatic shifts to almost the same degree as outdoors. Growth ofgenetically engineered plants in a greenhouse is quite costly. Incontrast, in the present system, every variable can be controlled sothat the maximum amount of expression can be achieved with everyharvest.

In certain embodiments, sprouted seedlings as provided herein can begrown in trays that can be watered, sprayed, or misted at any timeduring development of sprouted seedling. For example, a tray may befitted with one or more watering, spraying, misting, and drainingapparatus that can deliver and/or remove water, nutrients, and/orchemicals at specific time and at precise quantities during developmentof the sprouted seedling. For example, seeds require sufficient moistureto keep them damp. Excess moisture drains through holes in trays intodrains in the floor of the room. Typically, drainage water is treated asappropriate for removal of harmful chemicals before discharge back intothe environment.

Another advantage of trays is that they can be contained within a verysmall space. Since no light is required for sprouted seedlings to grow,trays containing seeds, embryos, or sprouted seedlings may be tightlystacked vertically on top of one another, providing a large quantity ofbiomass per unit floor space in a housing facility constructedspecifically for these purposes. In addition, stacks of trays can bearranged in horizontal rows within the housing unit. Once seedlings havegrown to a stage appropriate for harvest (about two to fourteen days)individual seedling trays are moved into a processing facility, eithermanually or by automatic means, such as a conveyor belt.

The system provided herein is unique in that it provides a sproutedseedling biomass, which is a source of a HA polypeptide(s). Whetherconsumed directly or processed into the form of a pharmaceuticalcomposition, because sprouted seedlings are grown in a contained,regulatable environment, sprouted seedling biomass and/or pharmaceuticalcomposition derived from biomass can be provided to a consumer at lowcost. In addition, the fact that the conditions for growth of sproutedseedlings can be controlled makes the quality and purity of productconsistent. A contained, regulatable environment can obviate many safetyregulations of the EPA that can prevent scientists from growinggenetically engineered agricultural products outdoors.

Transformed Sprouts

A variety of methods can be used to transform plant cells and producegenetically engineered sprouted seedlings. Two available methods fortransformation of plants that require that transgenic plant cell linesbe generated in vitro, followed by regeneration of cell lines into wholeplants include Agrobacterium tumefaciens mediated gene transfer andmicroprojectile bombardment or electroporation. In some embodiments,transient expression systems are utilized. Typical technologies forproducing transient expression of proteins or polypeptides in planttissues utilize plant viruses. Viral transformation provides more rapidand less costly methods of transforming embryos and sprouted seedlingsthat can be harvested without an experimental or generational lag priorto obtaining the desired product. For any of these techniques, theskilled artisan would appreciate how to adjust and optimizetransformation protocols that have traditionally been used for plants,seeds, embryos, or spouted seedlings.

This document provides expression systems having advantages of viralexpression systems (e.g., rapid expression, high levels of production)and of Agrobacterium transformation (e.g., controlled administration).In particular, as discussed in detail below, this document providessystems in which an agrobacterial construct (i.e., a construct thatreplicates in Agrobacterium and therefore can be delivered to plantcells by delivery of Agrobacterium) includes a plant promoter that,after being introduced into a plant, directs expression of viralsequences (e.g., including viral replication sequences) carrying a genefor a protein or polypeptide of interest. This system allows controlled,high level transient expression of proteins or polypeptides in plants.

A variety of different embodiments of expression systems, some of whichproduce transgenic plants and others of which provide for transientexpression, are discussed in further detail individually below. For anyof these techniques, the skilled artisan reading the presentspecification would appreciate how to adjust and optimize protocols forexpression of proteins or polypeptides in young plant tissues (e.g.,sprouted seedlings).

Agrobacterium Transformation

Agrobacterium is a representative genus of the gram-negative familyRhizobiaceae. This species is responsible for plant tumors such as crowngall and hairy root disease. In dedifferentiated plant tissue, which ischaracteristic of tumors, amino acid derivatives known as opines areproduced by the Agrobacterium and catabolized by the plant. Thebacterial genes responsible for expression of opines are a convenientsource of control elements for chimeric expression cassettes. In someembodiments, an Agrobacterium transformation system may be used togenerate young plants (e.g., sprouted seedlings, including ediblesprouted seedlings), which are merely harvested earlier than matureplants. Agrobacterium transformation methods can easily be applied toregenerate sprouted seedlings expressing HA polypeptides.

In general, transforming plants with Agrobacterium involvestransformation of plant cells grown in tissue culture by co-cultivationwith an Agrobacterium tumefaciens carrying a plant/bacterial vector. Thevector contains a gene encoding a HA polypeptide. The Agrobacteriumtransfers vector to plant host cell and is then eliminated usingantibiotic treatment. Transformed plant cells expressing HA polypeptideare selected, differentiated, and finally regenerated into completeplantlets (Hellens et al., 2000, Plant Mol. Biol., 42:819; Pilon-Smitset al., 1999, Plant Physiolog., 119:123; Barfield et al., 1991, PlantCell Reports, 10:308; and Riva et al., 1998, J. Biotech., 1(3); all ofwhich are incorporated by reference herein).

Agrobacterial expression vectors for use as described herein can includea gene (or expression cassette) encoding a HA polypeptide designed foroperation in plants, with companion sequences upstream and downstream ofthe expression cassette. Companion sequences are generally of plasmid orviral origin and provide necessary characteristics to the vector totransfer DNA from bacteria to the desired plant host.

The basic bacterial/plant vector construct may desirably provide a broadhost range prokaryote replication origin, a prokaryote selectablemarker. Suitable prokaryotic selectable markers include resistancetoward antibiotics such as ampicillin or tetracycline. Other DNAsequences encoding additional functions that are well known in the artmay be present in the vector.

Agrobacterium T-DNA sequences are required for Agrobacterium mediatedtransfer of DNA to the plant chromosome. The tumor-inducing genes ofT-DNA are typically removed during construction of an agrobacterialexpression construct and are replaced with sequences encoding a HApolypeptide. T-DNA border sequences are retained because they initiateintegration of the T-DNA region into the plant genome. If expression ofHA polypeptide is not readily amenable to detection, the bacterial/plantvector construct may include a selectable marker gene suitable fordetermining if a plant cell has been transformed, e.g., nptII kanamycinresistance gene. On the same or different bacterial/plant vector (Tiplasmid) are Ti sequences. Ti sequences include virulence genes, whichencode a set of proteins responsible for excision, transfer andintegration of T-DNA into the plant genome (Schell, 1987, Science,237:1176-86; incorporated herein by reference). Other sequences suitablefor permitting integration of heterologous sequence into the plantgenome may include transposon sequences, and the like, for homologousrecombination.

On the same or different bacterial/plant vector (Ti plasmid) are Tisequences. Ti sequences include the virulence genes, which encode a setof proteins responsible for the excision, transfer and integration ofthe T-DNA into the plant genome (Schell, 1987, Science, 237:1176-83;incorporated herein by reference). Other sequences suitable forpermitting integration of the heterologous sequence into the plantgenome may also include transposon sequences, and the like, forhomologous recombination.

Certain constructs will include an expression cassette encoding anantigen protein. One, two, or more expression cassettes may be used in agiven transformation. The recombinant expression cassette contains, inaddition to a HA polypeptide encoding sequence, at least the followingelements: a promoter region, plant 5′ untranslated sequences, initiationcodon (depending upon whether or not an expressed gene has its own), andtranscription and translation termination sequences. In addition,transcription and translation terminators may be included in expressioncassettes or chimeric genes. Signal secretion sequences that allowprocessing and translocation of a protein, as appropriate, may beincluded in the expression cassette.

A variety of promoters, signal sequences, and transcription andtranslation terminators are described, for example, in Lawton et al.(1987, Plant Mol. Biol., 9:315-24; incorporated herein by reference) orin U.S. Pat. No. 5,888,789 (incorporated herein by reference). Inaddition, structural genes for antibiotic resistance are commonlyutilized as a selection factor (Fraley et al., 1983, Proc. Natl. Acad.Sci., USA, 80:4803-7; incorporated herein by reference). Uniquerestriction enzyme sites at the 5′ and 3′ ends of the cassette allow foreasy insertion into a pre-existing vector.

Other binary vector systems for Agrobacterium-mediated transformation,carrying at least one T-DNA border sequence are described in PCTPublication WO 2000/020612 (incorporated herein by reference). Furtherdiscussion of Agrobacterium-mediated transformation is found in Gelvin(2003, Microbiol. Mol. Biol. Rev., 67:16-37; and references therein; allof which are incorporated herein by reference) and Lorence and Verpoorte(2004, Methods Mol. Biol., 267:329-50; incorporated herein byreference).

In certain embodiments, bacteria other than Agrobacteria are used tointroduce a nucleic acid sequence into a plant. See, e.g., Broothaertset al. (2005, Nature, 433:629-33; incorporated herein by reference).

Seeds are prepared from plants that have been infected with Agrobacteria(or other bacteria) such that the desired heterologous gene encoding aprotein or polypeptide of interest is introduced. Such seeds areharvested, dried, cleaned, and tested for viability and for the presenceand expression of a desired gene product. Once this has been determined,seed stock is typically stored under appropriate conditions oftemperature, humidity, sanitation, and security to be used whennecessary. Whole plants may then be regenerated from culturedprotoplasts, e.g., as described in Evans et al. (Handbook of Plant CellCultures, Vol. 1, MacMillan Publishing Co., New York, N.Y., 1983;incorporated herein by reference); and in Vasil (ed., Cell Culture andSomatic Cell Genetics of Plants, Acad. Press, Orlando, Fla., Vol. I,1984, and Vol. III, 1986; incorporated herein by reference). In certainaspects, plants are regenerated only to sprouted seedling stage. In someaspects, whole plants are regenerated to produce seed stocks andsprouted seedlings are generated from seeds of the seed stock.

In certain embodiments, the plants are not regenerated into adultplants. For example, in some embodiments, plants are regenerated only tothe sprouted seedling stage. In other embodiments, whole plants areregenerated to produce seed stocks and young plants (e.g., sproutedseedlings) for use in accordance with the present disclosure, and aregenerated from the seeds of previously produced seed stock.

All plants from which protoplasts can be isolated and cultured to givewhole, regenerated plants can be transformed by Agrobacteria so thatwhole plants are recovered that contain a transferred gene. It is knownthat practically all plants can be regenerated from cultured cells ortissues, including, but not limited to, all major species of plants thatproduce edible sprouts. Some suitable plants include alfalfa, mung bean,radish, wheat, mustard, spinach, carrot, beet, onion, garlic, celery,rhubarb, leafy plants such as cabbage and lettuce, watercress or cress,herbs such as parsley, mint, and clover, cauliflower, broccoli, soybean,lentils, and edible flowers such as sunflower.

Means for regeneration of plants from transformed cells vary from onespecies of plants to the next. However, those skilled in the art willappreciate that generally a suspension of transformed protoplantscontaining copies of a heterologous gene is first provided. Callustissue is formed and shoots may be induced from callus and subsequentlyrooted. Alternatively or additionally, embryo formation can be inducedfrom a protoplast suspension. These embryos germinate as natural embryosto form plants. Steeping seed in water or spraying seed with water toincrease the moisture content of the seed to between 35%-45% initiatesgermination. For germination to proceed, seeds are typically maintainedin air saturated with water under controlled temperature and airflowconditions. The culture media will generally contain various amino acidsand hormones, such as auxin and cytokinins. It is advantageous to addglutamic acid and proline to the medium, especially for such species asalfalfa. Shoots and roots normally develop simultaneously. Efficientregeneration will depend on the medium, the genotype, and the history ofthe culture. If these three variables are controlled, then regenerationis fully reproducible and repeatable.

Mature plants, grown from the transformed plant cells, are selfed andnon-segregating, homozygous transgenic plants are identified. The inbredplant produces seeds containing antigen-encoding sequences. Such seedscan be germinated and grown to sprouted seedling stage to produce HApolypeptide(s) as provided herein.

In related embodiments, transgenic seeds (e.g., carrying the transferredgene encoding a HA polypeptide, typically integrated into the genome)may be formed into seed products and sold with instructions on how togrow young plants to the appropriate stage (e.g., sprouted seedlingstage) for harvesting and/or administration or harvesting into aformulation as described herein. In some related embodiments, hybrids ornovel varieties embodying desired traits may be developed from inbredplants.

Direct Integration

Direct integration of DNA fragments into the genome of plant cells bymicroprojectile bombardment or electroporation may also be used tointroduce expression constructs encoding HA polypeptides into planttissues (see, e.g., Kikkert, et al., 1999, Plant: J. Tiss. Cult. Assoc.,35:43; and Bates, 1994, Mol. Biotech., 2:135; both of which areincorporated herein by reference). More particularly, vectors thatexpress HA polypeptide(s) can be introduced into plant cells by avariety of techniques. As described above, vectors may includeselectable markers for use in plant cells. Vectors may include sequencesthat allow their selection and propagation in a secondary host, such assequences containing an origin of replication and selectable marker.Typically, secondary hosts include bacteria and yeast. In someembodiments, a secondary host is bacteria (e.g., Escherichia coli, theorigin of replication is a colE1-type origin of replication) and aselectable marker is a gene encoding ampicillin resistance. Suchsequences are well known in the art and are commercially available(e.g., Clontech, Palo Alto, Calif. or Stratagene, La Jolla, Calif.).

Vectors as provided herein may be modified to intermediate planttransformation plasmids that contain a region of homology to anAgrobacterium tumefaciens vector, a T-DNA border region fromAgrobacterium tumefaciens, and chimeric genes or expression cassettesdescribed above. Further vectors may include a disarmed plant tumorinducing plasmid of Agrobacterium tumefaciens.

According to some embodiments, direct transformation of vectors caninvolve microinjecting vectors directly into plant cells by use ofmicropipettes to mechanically transfer recombinant DNA (see, e.g.,Crossway, 1985, Mol. Gen. Genet., 202:179, incorporated herein byreference). Genetic material may be transferred into a plant cell usingpolyethylene glycols (see, e.g., Krens et al., 1982, Nature 296:72;incorporated herein by reference). Another method of introducing nucleicacids into plants via high velocity ballistic penetration by smallparticles with a nucleic acid either within the matrix of small beads orparticles, or on the surface (see, e.g., Klein et al., 1987, Nature327:70; and Knudsen et al., Planta, 185:330; both of which areincorporated herein by reference). Yet another method of introduction isfusion of protoplasts with other entities, either minicells, cells,lysosomes, or other fusible lipid-surfaced bodies (see, e.g., Fraley etal., 1982, Proc. Natl. Acad. Sci., USA, 79:1859; incorporated herein byreference). Vectors in accordance with this document may be introducedinto plant cells by electroporation (see, e.g., Fromm et al. 1985, Proc.Natl. Acad. Sci., USA, 82:5824; incorporated herein by reference).According to this technique, plant protoplasts are electroporated in thepresence of plasmids containing a gene construct. Electrical impulses ofhigh field strength reversibly permeabilize biomembranes allowingintroduction of plasmids. Electroporated plant protoplasts reform thecell wall divide and form plant callus, which can be regenerated to formsprouted seedlings. Those skilled in the art will appreciate how toutilize these methods to transform plants cells that can be used togenerate edible sprouted seedlings.

Viral Transformation

Similar to conventional expression systems, plant viral vectors can beused to produce full-length proteins, including full length antigen.Plant virus vectors may be used to infect and produce antigen(s) inseeds, embryos, or sprouted seedlings, for example. In this regardinfection includes any method of introducing a viral genome, or portionthereof, into a cell, including, but not limited to, the naturalinfectious process of a virus, abrasion, and inoculation. The termincludes introducing a genomic RNA transcript, or a cDNA copy thereof,into a cell. The viral genome need not be a complete genome but willtypically contain sufficient sequences to allow replication. The genomemay encode a viral replicase and may contain any cis-acting nucleic acidelements necessary for replication. Expression of high levels of foreigngenes encoding short peptides as well as large complex proteins (e.g.,by tobamoviral vectors) is described (see, e.g., McCormick et al., 1999,Proc. Natl. Acad. Sci., USA, 96:703; Kumagai et al. 2000, Gene, 245:169;and Verch et al., 1998, J. Immunol. Methods, 220:69; all of which areincorporated herein by reference). Thus, plant viral vectors have ademonstrated ability to express short peptides as well as large complexproteins.

In certain embodiments, young plants (e.g., sprouts), which express HApolypeptide, are generated utilizing a host/virus system. Young plantsproduced by viral infection provide a source of transgenic protein thathas already been demonstrated to be safe. For example, sprouts are freeof contamination with animal pathogens. Unlike, for example, tobacco,proteins from an edible sprout could at least in theory be used in oralapplications without purification, thus significantly reducing costs.

In addition, a virus/young plant (e.g., sprout) system offers a muchsimpler, less expensive route for scale-up and manufacturing, since therelevant genes (encoding the protein or polypeptide of interest) areintroduced into the virus, which can be grown up to a commercial scalewithin a few days. In contrast, transgenic plants can require up to 5-7years before sufficient seeds or plant material is available forlarge-scale trials or commercialization.

As described herein, plant RNA viruses can have certain advantages,which make them attractive as vectors for foreign protein expression.The molecular biology and pathology of a number of plant RNA viruses arewell characterized and there is considerable knowledge of virus biology,genetics, and regulatory sequences. Most plant RNA viruses have smallgenomes and infectious cDNA clones are available to facilitate geneticmanipulation. Once infectious virus material enters a susceptible hostcell, it replicates to high levels and spreads rapidly throughout theentire sprouted seedling (one to ten days post inoculation, e.g., 1 day,2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,or more than 10 days post-inoculation). Virus particles are easily andeconomically recovered from infected sprouted seedling tissue. Viruseshave a wide host range, enabling use of a single construct for infectionof several susceptible species. These characteristics are readilytransferable to sprouts.

Foreign sequences can be expressed from plant RNA viruses, typically byreplacing one of the viral genes with desired sequence, by insertingforeign sequences into the virus genome at an appropriate position, orby fusing foreign peptides to structural proteins of a virus. Moreover,any of these approaches can be combined to express foreign sequences bytrans-complementation of vital functions of a virus. A number ofdifferent strategies exist as tools to express foreign sequences invirus-infected plants using tobacco mosaic virus (TMV), alfalfa mosaicvirus (AlMV), and chimeras thereof.

The genome of AlMV is a representative of the Bromoviridae family ofviruses and consists of three genomic RNAs (RNAs1-3) and subgenomic RNA(RNA4). Genomic RNAs1 and 2 encode virus replicase proteins P1 and 2,respectively. Genomic RNA3 encodes cell-to-cell movement protein P3 andcoat protein (CP). CP is translated from subgenomic RNA4, which issynthesized from genomic RNA3, and is required to start infection.Studies have demonstrated the involvement of CP in multiple functions,including genome activation, replication, RNA stability, symptomformation, and RNA encapsidation (see e.g., Bol et al., 1971, Virology,46:73; Van Der Vossen et al., 1994, Virology 202:891; Yusibov et al.,Virology, 208:405; Yusibov et al., 1998, Virology, 242:1; Bol et al.,(Review, 100 refs.), 1999, J. Gen. Virol., 80:1089; De Graaff, 1995,Virology, 208:583; Jaspars et al., 1974, Adv. Virus Res., 19:37;Loesch-Fries, 1985, Virology, 146:177; Neeleman et al., 1991, Virology,181:687; Neeleman et al., 1993, Virology, 196: 883; Van Der Kuyl et al.,1991, Virology, 183:731; and Van Der Kuyl et al., 1991, Virology,185:496; all of which are incorporated herein by reference).

Encapsidation of viral particles is typically required for long distancemovement of virus from inoculated to un-inoculated parts of seed,embryo, or sprouted seedling and for systemic infection. Inoculation canoccur at any stage of plant development. In embryos and sprouts, spreadof inoculated virus should be very rapid. Virions of AlMV areencapsidated by a unique CP (24 kD), forming more than one type ofparticle. The size (30- to 60-nm in length and 18 nm in diameter) andshape (spherical, ellipsoidal, or bacilliform) of the particle dependson the size of the encapsidated RNA. Upon assembly, the N-terminus ofAlMV CP is thought to be located on the surface of the virus particlesand does not appear to interfere with virus assembly (Bol et al., 1971,Virology, 6:73; incorporated herein by reference). Additionally, ALMV CPwith an additional 38-amino acid peptide at its N-terminus formsparticles in vitro and retains biological activity (Yusibov et al.,1995, J. Gen. Virol., 77:567; incorporated herein by reference).

AlMV has a wide host range, which includes a number of agriculturallyvaluable crop plants, including plant seeds, embryos, and sprouts.Together, these characteristics make ALMV CP an excellent candidate as acarrier molecule and AlMV an attractive candidate vector for expressionof foreign sequences in a plant at the sprout stage of development.Moreover, upon expression from a heterologous vector such as TMV, AlMVCP encapsidates TMV genome without interfering with virus infectivity(Yusibov et al., 1997, Proc. Natl. Acad. Sci., USA, 94:5784;incorporated herein by reference). This allows use of TMV as a carriervirus for AlMV CP fused to foreign sequences.

TMV, the prototype of tobamoviruses, has a genome consisting of a singleplus-sense RNA encapsidated with a 17.0 kD CP, which results inrod-shaped particles (300 nm in length). CP is the only structuralprotein of TMV and is required for encapsidation and long distancemovement of virus in an infected host (Saito et al., 1990, Virology176:329; incorporated herein by reference). 183 and 126 kD proteins aretranslated from genomic RNA and are required for virus replication(Ishikawa et al., 1986, Nucleic Acids Res., 14:8291; incorporated hereinby reference). 30 kD protein is the cell-to-cell movement protein ofvirus (Meshi et al., 1987, EMBO J., 6:2557). Movement and coat proteinsare translated from subgenomic mRNAs (Hunter et al., 1976, Nature,260:759; Bruening et al., 1976, Virology, 71:498; and Beachy et al.,1976, Virology, 73:498; all of which are incorporated herein byreference).

Other methods that may be utilized to introduce a gene encoding a HApolypeptide into plant cells include transforming the flower of a plant.Transformation of Arabidopsis thaliana can be achieved by dipping plantflowers into a solution of Agrobacterium tumefaciens (Curtis et al.,2001, Transgenic Res., 10:363; and Qing et al., 2000, MolecularBreeding: New Strategies in Plant Improvement 1:67; both of which areincorporated herein by reference). Transformed plants are formed in thepopulation of seeds generated by “dipped” plants. At a specific pointduring flower development, a pore exists in the ovary wall through whichAgrobacterium tumefaciens gains access to the interior of the ovary.Once inside the ovary, the Agrobacterium tumefaciens proliferates andtransforms individual ovules (Desfeux et al., 2000, Plant Physiology,123:895; incorporated herein by reference). Transformed ovules followthe typical pathway of seed formation within the ovary.

Agrobacterium-Mediated Transient Expression

As indicated herein, systems for rapid (e.g., transient) expression ofproteins or polypeptides in plants can be desirable. Among other things,this document provides a powerful system for achieving such rapidexpression in plants (particularly in young plants, e.g., sproutedseedlings) that utilizes an agrobacterial construct to deliver a viralexpression system encoding a HA polypeptide.

Specifically, as described herein, a “launch vector” is prepared thatcontains agrobacterial sequences including replication sequences andalso contains plant viral sequences (including self-replicationsequences) that carry a gene encoding the protein or polypeptide ofinterest. A launch vector is introduced into plant tissue, preferably byagroinfiltration, which allows substantially systemic delivery. Fortransient transformation, non-integrated T-DNA copies of the launchvector remain transiently present in the nucleolus and are transcribedleading to the expression of the carrying genes (Kapila et al., 1997,Plant Science, 122:101-108; incorporated herein by reference).Agrobacterium-mediated transient expression, differently from viralvectors, cannot lead to the systemic spreading of the expression of thegene of interest. One advantage of this system is the possibility toclone genes larger than 2 kb to generate constructs that would beimpossible to obtain with viral vectors (Voinnet et al., 2003, Plant 1,33:949-56; incorporated herein by reference). Furthermore, using suchtechnique, it is possible to transform the plant with more than onetransgene, such that multimeric proteins (e.g., antibodies subunits ofcomplexed proteins) can be expressed and assembled. Furthermore, thepossibility of co-expression of multiple transgenes by means ofco-infiltration with different Agrobacterium can be taken advantage of,either by separate infiltration or using mixed cultures.

In certain embodiments, a launch vector includes sequences that allowfor selection (or at least detection) in Agrobacteria and also forselection/detection in infiltrated tissues. Furthermore, a launch vectortypically includes sequences that are transcribed in the plant to yieldviral RNA production, followed by generation of viral proteins.Furthermore, production of viral proteins and viral RNA yields rapidproduction of multiple copies of RNA encoding the pharmaceuticallyactive protein of interest. Such production results in rapid proteinproduction of the target of interest in a relatively short period oftime. Thus, a highly efficient system for protein production can begenerated.

The agroinfiltration technique utilizing viral expression vectors can beused to produce limited quantity of protein of interest in order toverify the expression levels before deciding if it is worth generatingtransgenic plants. Alternatively or additionally, the agroinfiltrationtechnique utilizing viral expression vectors is useful for rapidgeneration of plants capable of producing huge amounts of protein as aprimary production platform. Thus, this transient expression system canbe used on industrial scale.

Further provided are any of a variety of different Agrobacterialplasmids, binary plasmids, or derivatives thereof such as pBIV, pBI1221,and pGreen, which can be used in these and other aspects of the presentdisclosure. Numerous suitable vectors are known in the art and can bedirected and/or modified according to methods known in the art, or thosedescribed herein so as to utilize in the methods described providedherein.

An exemplary launch vector, pBID4, contains the 35S promoter ofcauliflower mosaic virus (a DNA plant virus) that drives initialtranscription of the recombinant viral genome following introductioninto plants, and the nos terminator, the transcriptional terminator ofAgrobacterium nopaline synthase. The vector further contains sequencesof the tobacco mosaic virus genome including genes for virus replication(126/183K) and cell-t-cell movement (MP). The vector further contains agene encoding a polypeptide of interest, inserted into a unique cloningsite within the tobacco mosaic virus genome sequences and under thetranscriptional control of the coat protein subgenomic mRNA promoter.Because this “target gene” (i.e., gene encoding a protein or polypeptideof interest) replaces coding sequences for the TMV coat protein, theresultant viral vector is naked self-replicating RNA that is lesssubject to recombination than CP-containing vectors, and that cannoteffectively spread and survive in the environment. Left and right bordersequences (LB and RB) delimit the region of the launch vector that istransferred into plant cells following infiltration of plants withrecombinant Agrobacterium carrying the vector. Upon introduction ofagrobacteria carrying this vector into plant tissue (typically byagroinfiltration but alternatively by injection or other means),multiple single-stranded DNA (ssDNA) copies of sequence between LB andRB are generated and released in a matter of minutes. These introducedsequences are then amplified by viral replication. Translation of thetarget gene results in accumulation of large amounts of target proteinor polypeptide in a short period of time.

In some embodiments, Agrobacterium-mediated transient expressionproduces up to about 5 g or more of target protein per kg of planttissue. For example, in some embodiments, up to about 4 g, about 3 g,about 2 g, about 1 g, or about 0.5 g of target protein is produced perkg of plant tissue. In some embodiments, at least about 20 mg to about500 mg, or about 50 mg to about 500 mg of target protein, or about 50 mgto about 200 mg, or about 50 mg, about 60 mg, about 70 mg, about 80 mg,about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg,about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg,about 190 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg,about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg,about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg,about 900 mg, about 950 mg, about 1000 mg, about 1500 mg, about 1750 mg,about 2000 mg, about 2500 mg, about 3000 mg or more of protein per kg ofplant tissue is produced.

In some embodiments, these expression levels are achieved within about6, about 5, about 4, about 3, or about 2 weeks from infiltration. Insome embodiments, these expression levels are achieved within about 10,about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2days, or even about 1 day, from introduction of the expressionconstruct. Thus, the time from introduction (e.g., infiltration) toharvest is typically less than about 2 weeks, about 10 days, about 1week or less. This allows production of protein within about 8 weeks orless from the selection of amino acid sequence (even including time for“preliminary” expression studies). Also, each batch of protein cantypically be produced within about 8 weeks, about 6 weeks, about 5weeks, or less. Those of ordinary skill in the art will appreciate thatthese numbers may vary somewhat depending on the type of plant used.Most sprouts, including peas, will fall within the numbers given.Nicotiana benthamiana, however, may be grown longer, particularly priorto infiltration, as they are slower growing (from a much smaller seed).Other expected adjustments will be clear to those of ordinary skill inthe art based on biology of the particular plants utilized.

A launch vector system has been used to produce a variety of targetproteins and polypeptides in a variety of different young plants. Insome embodiments, certain pea varieties including for example, marrowfatpea, bill jump pea, yellow trapper pea, speckled pea, and green pea areparticularly useful in the practice of methods as disclosed herein, forexample.

Various Nicotiana plants can be particularly useful in the practice ofthis disclosure, including in particular Nicotiana benthamiana. It willbe understood by those of ordinary skill in the art that Nicotianaplants are generally not considered to be “sprouts.” Nonetheless, youngNicotiana plants (particularly young Nicotiana benthamiana plants) canbe useful in the practices provided herein. In general, in someembodiments, Nicotiana benthamiana plants are grown for a timesufficient to allow development of an appropriate amount of biomassprior to infiltration (i.e., to delivery of agrobacteria containing thelaunch vector). Typically, the plants are grown for a period of morethan about 3 weeks, more typically more than about 4 weeks, or betweenabout 5 to about 6 weeks to accumulate biomass prior to infiltration.

It has further surprisingly been found that, although both TMV and AlMVsequences can prove effective in such launch vector constructs, in someembodiments, AlMV sequences are particularly efficient at ensuring highlevel production of delivered protein or polypeptides.

Thus, in some embodiments, proteins or polypeptides of interest can beproduced in young pea plants or young Nicotania plants (e.g., Nicotianabenthamiana) from a launch vector that directs production of AlMVsequences carrying the gene of interest.

Expression Constructs

Many features of expression constructs useful as described herein willbe specific to the particular expression system used, as discussedabove. However, certain aspects that may be applicable across differentexpression systems are discussed in further detail here.

To give but one example, in some embodiments, it will be desirable thatexpression of the protein or polypeptide (or nucleic acid) of interestbe inducible. In many such embodiments, production of an RNA encodingthe protein or polypeptide of interest (and/or production of anantisense RNA) is under the control of an inducible (e.g. exogenouslyinducible) promoter. Exogenously inducible promoters are caused toincrease or decrease expression of a transcript in response to anexternal, rather than an internal stimulus. A number of environmentalfactors can act as such an external stimulus. In certain embodiments,transcription is controlled by a heat-inducible promoter, such as aheat-shock promoter.

Externally inducible promoters may be particularly useful in the contextof controlled, regulatable growth settings. For example, using aheat-shock promoter the temperature of a contained environment maysimply be raised to induce expression of the relevant transcript. Inwill be appreciated, of course, that a heat inducible promoter couldnever be used in the outdoors because the outdoor temperature cannot becontrolled. The promoter would be turned on any time the outdoortemperature rose above a certain level. Similarly, the promoter would beturned off every time the outdoor temperature dropped. Such temperatureshifts could occur in a single day, for example, turning expression onin the daytime and off at night. A heat inducible promoter, such asthose described herein, would likely not even be practical for use in agreenhouse, which is susceptible to climatic shifts to almost the samedegree as the outdoors. Growth of genetically engineered plants in agreenhouse is quite costly. In contrast, in the present system, everyvariable can be controlled so that the maximum amount of expression canbe achieved with every harvest.

Other externally-inducible promoters than can be utilized include lightinducible promoters. Light-inducible promoters can be maintained asconstitutive promoters if the light in the contained regulatableenvironment is always on. Alternatively, expression of the relevanttranscript can be turned on at a particular time during development bysimply turning on the light.

In yet other embodiments, a chemically inducible promoter is used toinduce expression of the relevant transcript. According to theseembodiments, the chemical could simply be misted or sprayed onto a seed,embryo, or young plant (e.g., seedling) to induce expression of therelevant transcript. Spraying and misting can be precisely controlledand directed onto a particular seed, embryo, or young plant (e.g.,seedling) as desired. A contained environment is devoid of wind or aircurrents, which could disperse the chemical away from the intendedrecipient, so that the chemical stays on the recipient for which it wasintended.

Production and Isolation of Antigen

In general, standard methods known in the art may be used for culturingor growing plants, plant cells, and/or plant tissues (e.g., clonalplants, clonal plant cells, clonal roots, clonal root lines, sprouts,sprouted seedlings, and plants) for production of antigen(s). A widevariety of culture media and bioreactors have been employed to culturehairy root cells, root cell lines, and plant cells (see, for example,Giri et al., 2000, Biotechnol. Adv., 18:1; Rao et al., 2002, Biotechnol.Adv., 20:101; and references in both of the foregoing, all of which areincorporated herein by reference). Clonal plants may be grown in anysuitable manner.

In a certain embodiments, HA polypeptides as provided herein can beproduced by any known method. In some embodiments, a HA polypeptide isexpressed in a plant or portion thereof. Proteins are isolated andpurified in accordance with conventional conditions and techniques knownin the art. These include methods such as extraction, precipitation,chromatography, affinity chromatography, electrophoresis, and the like.Thus, this document provides methods that include purification andaffordable scaling up of production of HA polypeptide(s) using any of avariety of plant expression systems known in the art and providedherein, including viral plant expression systems described herein.

In some embodiments, it can be desirable to isolate HA polypeptide(s)for vaccine products. Where a protein is produced from plant tissue(s)or a portion thereof, e.g., roots, root cells, plants, plant cells, thatexpress them, methods described in further detail herein, or anyapplicable methods known in the art may be used for any of partial orcomplete isolation from plant material. Where it is desirable to isolatethe expression product from some or all of plant cells or tissues thatexpress it, any available purification techniques may be employed. Thoseof ordinary skill in the art are familiar with a wide range offractionation and separation procedures (see, for example, Scopes etal., Protein Purification: Principles and Practice, 3^(rd) Ed., Jansonet al., 1993; Protein Purification: Principles, High Resolution Methods,and Applications, Wiley-VCH, 1998; Springer-Verlag, NY, 1993; and Roe,Protein Purification Techniques, Oxford University Press, 2001; each ofwhich is incorporated herein by reference). Often, it will be desirableto render the product more than about 50%, about about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% pure. See, e.g., U.S. Pat.Nos. 6,740,740 and 6,841,659 (both of which are incorporated herein byreference) for discussion of certain methods useful for purifyingsubstances from plant tissues or fluids.

Those skilled in the art will appreciate that a method of obtainingdesired HA polypeptide(s) product(s) is by extraction. Plant material(e.g., roots and/or leaves) may be extracted to remove desired productsfrom residual biomass, thereby increasing the concentration and purityof product. Plants may be extracted in a buffered solution. For example,plant material may be transferred into an amount of ice-cold water at aratio of one to one by weight that has been buffered with, e.g.,phosphate buffer. Protease inhibitors can be added as required. Theplant material can be disrupted by vigorous blending or grinding whilesuspended in buffer solution and extracted biomass removed by filtrationor centrifugation. The product carried in solution can be furtherpurified by additional steps or converted to a dry powder byfreeze-drying or precipitation. Extraction can be carried out bypressing. Plants or roots can be extracted by pressing in a press or bybeing crushed as they are passed through closely spaced rollers. Fluidsexpressed from crushed plants or roots are collected and processedaccording to methods well known in the art. Extraction by pressingallows release of products in a more concentrated form. However, overallyield of product may be lower than if product were extracted insolution.

In some embodiments, produced proteins or polypeptides are not isolatedfrom plant tissue but rather are provided in the context of live plants(e.g., sprouted seedlings). In some embodiments, where the plant isedible, plant tissue containing expressed protein or polypeptide isprovided directly for consumption. Thus, this document provides edibleyoung plant biomass (e.g., edible sprouted seedlings) containingexpressed protein or polypeptide.

Where edible plants (e.g., sprouted seedlings) express sufficient levelsof pharmaceutical proteins or polypeptides and are consumed live, insome embodiments absolutely no harvesting occurs before the sproutedseedlings are consumed. In this way, it is guaranteed that there is noharvest-induced proteolytic breakdown of the pharmaceutical proteinbefore administration of the pharmaceutical protein to a patient in needof treatment. For example, young plants (e.g., sprouted seedlings) thatare ready to be consumed can be delivered directly to a patient.Alternatively, genetically engineered seeds or embryos are delivered toa patient in need of treatment and grown to the sprouted seedling stageby the patient. In some embodiments, a supply of genetically engineeredsprouted seedlings is provided to a patient, or to a doctor who will betreating patients, so that a continual stock of sprouted seedlingsexpressing certain desirable pharmaceutical proteins may be cultivated.This may be particularly valuable for populations in developingcountries, where expensive pharmaceuticals are not affordable ordeliverable. The ease with which the sprouted seedlings can be grown canmake them particularly desirable for such developing populations.

In some embodiments, plant biomass is processed prior to consumption orformulation, for example, by homogenizing, crushing, drying, orextracting. In some embodiments, the expressed protein or polypeptide isisolated or purified from the biomass and formulated into apharmaceutical composition.

For example, live plants (e.g., sprouts) may be ground, crushed, orblended to produce a slurry of biomass, in a buffer containing proteaseinhibitors. Preferably the buffer is at about 4° C. In certainembodiments, the biomass is air-dried, spray dried, frozen, orfreeze-dried. As in mature plants, some of these methods, such asair-drying, may result in a loss of activity of the pharmaceuticalprotein or polypeptide. However, because plants (e.g., sproutedseedlings) may be very small and typically have a large surface area tovolume ratio, this is much less likely to occur. Those skilled in theart will appreciate that many techniques for harvesting the biomass thatminimize proteolysis of the pharmaceutical protein or polypeptide areavailable and could be applied to the methods provided herein.

Antibodies

This document also provides pharmaceutical antigen and antibody proteinsfor therapeutic use, such as influenza antigen(s) (e.g., influenzaprotein(s) or an immunogenic portion(s) thereof, or fusion proteinscomprising influenza antibody protein(s) or an antigen bindingportion(s) thereof) active as antibody for therapeutic and/orprophylactic treatment of influenza infection. Further, this documentprovides veterinary uses, as such influenza antigen is active inveterinary applications. In certain embodiments, influenza antigen(s)and/or antibodies may be produced by plant(s) or portion(s) thereof(e.g., root, cell, sprout, cell line, or plant) as described herein. Incertain embodiments, provided influenza antigens and/or antibodies areexpressed in plants, plant cells, and/or plant tissues (e.g., sprouts,sprouted seedlings, roots, root culture, clonal cells, clonal celllines, or clonal plants), and can be used directly from plant orpartially purified or purified in preparation for pharmaceuticaladministration to a subject.

Monoclonal Antibodies

Various methods for generating monoclonal antibodies (MAbs) are now verywell known in the art. The most standard monoclonal antibody generationtechniques generally begin along the same lines as those for preparingpolyclonal antibodies (Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988, which is hereby incorporated by reference). Apolyclonal antibody response is initiated by immunizing an animal withan immunogenic anionic phospholipid and/or aminophospholipid compositionand, when a desired titer level is obtained, the immunized animal can beused to generate MAbs. Typically, the particular screening and selectiontechniques disclosed herein are used to select antibodies with thesought after properties.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, the technique involves immunizing a suitableanimal with a selected immunogen composition to stimulate antibodyproducing cells. Rodents such as mice and rats are exemplary animals,however, the use of rabbit, sheep and frog cells is possible. The use ofrats may provide certain advantages (Goding, 1986, pp. 60-61;incorporated herein by reference), but mice are sometimes preferred,with the BALB/c mouse often being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

Following immunization, somatic cells with the potential for producingthe desired antibodies, specifically B lymphocytes (B cells), areselected for use in the MAb generation and fusion with cells of animmortal myeloma cell, generally one of the same species as the animalthat was immunized. Myeloma cell lines suited for use inhybridoma-producing fusion procedures typically arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas). Any one of a number of myeloma cells may be used, as areknown to those of skill in the art (Goding, pp. 65-66, 1986; Campbell,pp. 75-83, 1984; each incorporated herein by reference). For example,where the immunized animal is a mouse, one may use P3-X63/Ag8,X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11—X45-GTG1.7 and 5194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3,IR983F, 4B210 or one of the above listed mouse cell lines; and U-266,GM1500-GRG2, LICR-LON-HMy2 and UC729-6, are all useful in connectionwith human cell fusions.

This culturing provides a population of hybridomas from which specifichybridomas are selected, followed by serial dilution and cloning intoindividual antibody producing lines, which can be propagatedindefinitely for production of antibody.

MAbs produced are generally be further purified, e.g., using filtration,centrifugation and various chromatographic methods, such as HPLC oraffinity chromatography, all of which purification techniques are wellknown to those of skill in the art. These purification techniques eachinvolve fractionation to separate the desired antibody from othercomponents of a mixture. Analytical methods particularly suited to thepreparation of antibodies include, for example, protein A-Sepharoseand/or protein G-Sepharose chromatography.

Antibody Fragments and Derivatives

Irrespective of the source of the original antibody against ahemagglutinin, either the intact antibody, antibody multimers, or anyone of a variety of functional, antigen-binding regions of the antibodymay be used. Exemplary functional regions include scFv, Fv, Fab′, Faband F(ab′).sub.2 fragments of antibodies. Techniques for preparing suchconstructs are well known to those in the art and are furtherexemplified herein.

The choice of antibody construct may be influenced by various factors.For example, prolonged half-life can result from the active readsorptionof intact antibodies within the kidney, a property of the Fc piece ofimmunoglobulin. IgG based antibodies, therefore, are expected to exhibitslower blood clearance than their Fab′ counterparts. However, Fab′fragment-based compositions will generally exhibit better tissuepenetrating capability.

Antibody fragments can be obtained by proteolysis of the wholeimmunoglobulin by the non-specific thiolprotease, papain. Papaindigestion yields two identical antigen-binding fragments, termed “Fabfragments,” each with a single antigen-binding site, and a residual “Fcfragment.” The various fractions are separated by protein A-Sepharose orion exchange chromatography.

The usual procedure for preparation of F(ab′).sub.2 fragments from IgGof rabbit and human origin is limited proteolysis by the enzyme pepsin.Pepsin treatment of intact antibodies yields an F(ab′).sub.2 fragmentthat has two antigen-combining sites and is still capable ofcross-linking antigen.

A Fab fragment contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH1 domain including one or more cysteine(s)from the antibody hinge region. F(ab′).sub.2 antibody fragments wereoriginally produced as pairs of Fab′ fragments that have hinge cysteinesbetween them. Other chemical couplings of antibody fragments are known.

An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen-recognition and binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,con-covalent association. It is in this configuration that threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)—V_(L) dimerCollectively, six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

“Single-chain Fv” or “scFv” antibody fragments (now known as “singlechains”) comprise the V_(H) and V_(L) domains of an antibody, whereinthese domains are present in a single polypeptide chain. Generally, theFv polypeptide further comprises a polypeptide linker between V_(H) andV_(L) domains that enables sFv to form the desired structure for antigenbinding.

The following patents are incorporated herein by reference for thepurposes of even further supplementing the present teachings regardingthe preparation and use of functional, antigen-binding regions ofantibodies, including scFv, Fv, Fab′, Fab and F(ab′).sub.2 fragments ofantibodies: U.S. Pat. Nos. 5,855,866; 5,877,289; 5,965,132; 6,093,399;6,261,535; and 6,004,555. WO 98/45331 is also incorporated herein byreference for purposes including even further describing and teachingthe preparation of variable, hypervariable and complementaritydetermining (CDR) regions of antibodies.

“Diabodies” are small antibody fragments with two antigen-binding sites,which fragments comprise a heavy chain variable domain (V_(H)) connectedto a light chain variable domain (V_(L)) in the same polypeptide chain(V_(H)—V_(L)). By using a linker that is too short to allow pairingbetween two domains on the same chain, the domains are forced to pairwith the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described in EP 404,097 and WO93/11161, each specifically incorporated herein by reference. “Linearantibodies,” which can be bispecific or monospecific, comprise a pair oftandem Fd segments (V.sub.H—C.sub.H1-V.sub.H—C.sub.H1) that form a pairof antigen binding regions, as described (see, for example, Zapata etal., 1995, incorporated herein by reference).

In using a Fab′ or antigen binding fragment of an antibody, with theattendant benefits on tissue penetration, one may derive additionaladvantages from modifying the fragment to increase its half-life. Avariety of techniques may be employed, such as manipulation ormodification of the antibody molecule itself, and conjugation to inertcarriers. Any conjugation for the sole purpose of increasing half-life,rather than to deliver an agent to a target, should be approachedcarefully in that Fab′ and other fragments are chosen to penetratetissues. Nonetheless, conjugation to non-protein polymers, such PEG andthe like, is contemplated.

Modifications other than conjugation are therefore based upon modifyingthe structure of the antibody fragment to render it more stable, and/orto reduce the rate of catabolism in the body. One mechanism for suchmodifications is the use of D-amino acids in place of L-amino acids.Those of ordinary skill in the art will understand that the introductionof such modifications needs to be followed by rigorous testing of theresultant molecule to ensure that it still retains the desiredbiological properties. Further stabilizing modifications include the useof the addition of stabilizing moieties to either N-terminal orC-terminal, or both, which is generally used to prolong half-life ofbiological molecules. By way of example only, one may wish to modifytermini by acylation or amination.

Bispecific Antibodies

Bispecific antibodies in general may be employed, so long as one armbinds to an aminophospholipid or anionic phospholipid and the bispecificantibody is attached, at a site distinct from the antigen binding site,to a therapeutic agent.

In general, the preparation of bispecific antibodies is well known inthe art. One method involves the separate preparation of antibodieshaving specificity for the aminophospholipid or anionic phospholipid, onthe one hand, and a therapeutic agent on the other. Peptic F(ab′)₂fragments are prepared from two chosen antibodies, followed by reductionof each to provide separate Fab′_(SH) fragments. SH groups on one of twopartners to be coupled are then alkylated with a cross-linking reagentsuch as O-phenylenedimaleimide to provide free maleimide groups on onepartner. This partner may then be conjugated to the other by means of athioether linkage, to give the desired F(ab′)₂ heteroconjugate. Othertechniques are known wherein cross-linking with SPDP or protein A iscarried out, or a trispecific construct is prepared.

One method for producing bispecific antibodies is by the fusion of twohybridomas to form a quadroma. As used herein, the term “quadroma” isused to describe the productive fusion of two B cell hybridomas. Usingnow standard techniques, two antibody producing hybridomas are fused togive daughter cells, and those cells that have maintained the expressionof both sets of clonotype immunoglobulin genes are then selected.

CDR Technologies

Antibodies are comprised of variable and constant regions. The term“variable,” as used herein in reference to antibodies, means thatcertain portions of the variable domains differ extensively in sequenceamong antibodies, and are used in the binding and specificity of eachparticular antibody to its particular antigen. However, the variabilityis concentrated in three segments termed “hypervariable regions,” bothin the light chain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called theframework region (FR). Variable domains of native heavy and light chainseach comprise four FRs (FR1, FR2, FR3 and FR4, respectively), largelyadopting a beta-sheet configuration connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the beta-sheet structure.

The hypervariable regions in each chain are held together in closeproximity by the FRs and, with hypervariable regions from the otherchain, contribute to the formation of the antigen-binding site ofantibodies (Kabat et al., 1991, incorporated herein by reference).Constant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody-dependent cellular toxicity.

The term “hypervariable region,” as used herein, refers to amino acidresidues of an antibody that are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-56 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., 1991, incorporated herein by reference) and/or thoseresidues from a “hypervariable loop” (i.e. residues 26-32 (L1),50-52(L2) and 91-96 (L3) in the light chain variable domain and 26-32(H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain).“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

The DNA and deduced amino acid sequences of Vh and V kappa chains of theHA antibodies described in FIGS. 9 and 10 encompass CDR1-3 of variableregions of heavy and light chains of the antibody. In light of thesequence and other information provided herein, and the knowledge in theart, a range of antibodies similar to those described in FIGS. 9 and 10,and improved antibodies and antigen binding regions, can now be preparedand are thus encompassed by this disclosure. Sequences of the light andheavy chain variable regions of the HA antibodies described in FIGS. 9and 10 can be determined using standard techniques.

In certain embodiments, this document provides at least one CDR of theantibody produced by one or more of the HA antibodies described in FIGS.9 and 10, to be deposited. In some embodiments, this document provides aCDR, antibody, or antigen binding region thereof, which binds to atleast a hemagglutinin, and which comprises at least one CDR of theantibody produced by one or more of the HA antibodies described in FIGS.9 and 10, to be deposited.

In one embodiment, this document provides an antibody, or antigenbinding region thereof, in which the framework regions of one or more ofthe HA antibodies described in FIGS. 9 and 10 have been changed frommouse to a human IgG, such as human IgG1 or other IgG subclass to reduceimmunogenicity in humans. In some embodiments, sequences of one or moreof the HA antibodies described in FIGS. 9 and 10 are examined for thepresence of T-cell epitopes, as is known in the art. The underlyingsequence can then be changed to remove T-cell epitopes, i.e., to“deimmunize” the antibody.

The availability of DNA and amino acid sequences of Vh and V kappachains of one or more of the HA antibodies described in FIGS. 9 and 10means that a range of antibodies can now be prepared using CDRtechnologies. In particular, random mutations are made in the CDRs andproducts screened to identify antibodies with higher affinities and/orhigher specificities. Such mutagenesis and selection is routinelypracticed in the antibody arts, and it can be particularly suitable foruse in the methods provided herein, given the advantageous screeningtechniques disclosed herein. A convenient way for generating suchsubstitutional variants is affinity maturation using phage display.

CDR shuffling and implantation technologies can be used with antibodiesin accordance with the present disclosure, specifically one or more ofthe HA antibodies described in FIGS. 9 and 10. CDR shuffling inserts CDRsequences into a specific framework region (Jirholt et al., 1998,incorporated herein by reference). CDR implantation techniques permitrandom combination of CDR sequences into a single master framework(Soderlind et al., 1999, 2000, each incorporated herein by reference).Using such techniques, CDR sequences of one or more of the HA antibodiesdescribed in FIGS. 9 and 10, for example, are mutagenized to create aplurality of different sequences, which are incorporated into a scaffoldsequence and the resultant antibody variants screened for desiredcharacteristics, e.g., higher affinity.

Antibodies from Phagemid Libraries

Recombinant technology now allows the preparation of antibodies having adesired specificity from recombinant genes encoding a range ofantibodies (Van Dijk et al., 1989; incorporated herein by reference).Certain recombinant techniques involve isolation of antibody genes byimmunological screening of combinatorial immunoglobulin phage expressionlibraries prepared from RNA isolated from spleen of an immunized animal(Morrison et al., 1986; Winter and Milstein, 1991; Barbas et al., 1992;each incorporated herein by reference). For such methods, combinatorialimmunoglobulin phagemid libraries are prepared from RNA isolated fromspleen of an immunized animal, and phagemids expressing appropriateantibodies are selected by panning using cells expressing antigen andcontrol cells. Advantage of this approach over conventional hybridomatechniques include approximately 10⁴ times as many antibodies can beproduced and screened in a single round, and that new specificities aregenerated by H and L chain combination, which further increases thepercentage of appropriate antibodies generated.

One method for the generation of a large repertoire of diverse antibodymolecules in bacteria utilizes the bacteriophage lambda as the vector(Huse et al., 1989; incorporated herein by reference). Production ofantibodies using the lambda vector involves the cloning of heavy andlight chain populations of DNA sequences into separate starting vectors.Vectors are subsequently combined randomly to form a single vector thatdirects co-expression of heavy and light chains to form antibodyfragments. The general technique for filamentous phage display isdescribed (U.S. Pat. No. 5,658,727, incorporated herein by reference).In a most general sense, the method provides a system for thesimultaneous cloning and screening of pre-selected ligand-bindingspecificities from antibody gene repertoires using a single vectorsystem. Screening of isolated members of the library for a pre-selectedligand-binding capacity allows the correlation of the binding capacityof an expressed antibody molecule with a convenient means to isolate agene that encodes the member from the library. Additional methods forscreening phagemid libraries are described (U.S. Pat. Nos. 5,580,717;5,427,908; 5,403,484; and 5,223,409, each incorporated herein byreference).

One method for the generation and screening of large libraries of whollyor partially synthetic antibody combining sites, or paratopes, utilizesdisplay vectors derived from filamentous phage such as M13, fl or fd(U.S. Pat. No. 5,698,426, incorporated herein by reference). Filamentousphage display vectors, referred to as “phagemids,” yield large librariesof monoclonal antibodies having diverse and novel immunospecificities.The technology uses a filamentous phage coat protein membrane anchordomain as a means for linking gene-product and gene during the assemblystage of filamentous phage replication, and has been used for thecloning and expression of antibodies from combinatorial libraries (Kanget al., 1991; Barbas et al., 1991; each incorporated herein byreference). The surface expression library is screened for specific Fabfragments that bind hemagglutinin molecules by standard affinityisolation procedures. The selected Fab fragments can be characterized bysequencing the nucleic acids encoding the polypeptides afteramplification of the phage population.

One method for producing diverse libraries of antibodies and screeningfor desirable binding specificities is described (U.S. Pat. Nos.5,667,988 and 5,759,817, each incorporated herein by reference). Themethod involves the preparation of libraries of heterodimericimmunoglobulin molecules in the form of phagemid libraries usingdegenerate oligonucleotides and primer extension reactions toincorporate degeneracies into CDR regions of immunoglobulin variableheavy and light chain variable domains, and display of mutagenizedpolypeptides on the surface of the phagemid. Thereafter, the displayprotein is screened for the ability to bind to a preselected antigen. Afurther variation of this method for producing diverse libraries ofantibodies and screening for desirable binding specificities isdescribed U.S. Pat. No. 5,702,892, incorporated herein by reference). Inthis method, only heavy chain sequences are employed, heavy chainsequences are randomized at all nucleotide positions which encode eitherthe CDRI or CDRIII hypervariable region, and the genetic variability inthe CDRs is generated independent of any biological process.

Transgenic Mice Containing Human Antibody Libraries

Recombinant technology is available for the preparation of antibodies.In addition to the combinatorial immunoglobulin phage expressionlibraries disclosed above, one molecular cloning approach is to prepareantibodies from transgenic mice containing human antibody libraries.Such techniques are described (U.S. Pat. No. 5,545,807, incorporatedherein by reference).

In a most general sense, these methods involve the production of atransgenic animal that has inserted into its germline genetic materialthat encodes for at least part of an immunoglobulin of human origin orthat can rearrange to encode a repertoire of immunoglobulins. Theinserted genetic material may be produced from a human source, or may beproduced synthetically. The material may code for at least part of aknown immunoglobulin or may be modified to code for at least part of analtered immunoglobulin.

The inserted genetic material is expressed in the transgenic animal,resulting in production of an immunoglobulin derived at least in partfrom the inserted human immunoglobulin genetic material. The insertedgenetic material may be in the form of DNA cloned into prokaryoticvectors such as plasmids and/or cosmids. Larger DNA fragments areinserted using yeast artificial chromosome vectors (Burke et al., 1987;incorporated herein by reference), or by introduction of chromosomefragments (Richer et al., 1989; incorporated herein by reference). Theinserted genetic material may be introduced to the host in conventionalmanner, for example by injection or other procedures into fertilizedeggs or embryonic stem cells.

Once a suitable transgenic animal has been prepared, the animal issimply immunized with the desired immunogen. Depending on the nature ofthe inserted material, the animal may produce a chimeric immunoglobulin,e.g. of mixed mouse/human origin, where the genetic material of foreignorigin encodes only part of the immunoglobulin; or the animal mayproduce an entirely foreign immunoglobulin, e.g. of wholly human origin,where the genetic material of foreign origin encodes an entireimmunoglobulin.

Polyclonal antisera may be produced from the transgenic animal followingimmunization. Immunoglobulin-producing cells may be removed from theanimal to produce the immunoglobulin of interest. Generally, monoclonalantibodies are produced from the transgenic animal, e.g., by fusingspleen cells from the animal with myeloma cells and screening theresulting hybridomas to select those producing the desired antibody.Suitable techniques for such processes are described herein.

In one approach, the genetic material may be incorporated in the animalin such a way that the desired antibody is produced in body fluids suchas serum or external secretions of the animal, such as milk, colostrumor saliva. For example, by inserting in vitro genetic material encodingfor at least part of a human immunoglobulin into a gene of a mammalcoding for a milk protein and then introducing the gene to a fertilizedegg of the mammal, e.g., by injection, the egg may develop into an adultfemale mammal producing milk containing immunoglobulin derived at leastin part from the inserted human immunoglobulin genetic material. Thedesired antibody can then be harvested from the milk. Suitabletechniques for carrying out such processes are known to those skilled inthe art.

The foregoing transgenic animals are usually employed to produce humanantibodies of a single isotype, more specifically an isotype that isessential for B cell maturation, such as IgM and possibly IgD. Anothermethod for producing human antibodies is described in U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,770,429;each incorporated by reference, wherein transgenic animals are describedthat are capable of switching from an isotype needed for B celldevelopment to other isotypes.

In the method described in U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,661,016; and 5,770,429, human immunoglobulintransgenes contained within a transgenic animal function correctlythroughout the pathway of B-cell development, leading to isotypeswitching. Accordingly, in this method, these transgenes are constructedso as to produce isotype switching and one or more of the following: (1)high level and cell-type specific expression, (2) functional generearrangement, (3) activation of and response to allelic exclusion, (4)expression of a sufficient primary repertoire, (5) signal transduction,(6) somatic hypermutation, and (7) domination of the transgene antibodylocus during the immune response.

Humanized Antibodies

Human antibodies generally have at least three potential advantages foruse in human therapy. First, because the effector portion is human, itmay interact better with other parts of the human immune system, e.g.,to destroy target cells more efficiently by complement-dependentcytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC).Second, the human immune system should not recognize the antibody asforeign. Third, half-life in human circulation will be similar tonaturally occurring human antibodies, allowing smaller and less frequentdoses to be given.

Various methods for preparing human antibodies are provided herein. Inaddition to human antibodies, “humanized” antibodies have manyadvantages. “Humanized” antibodies are generally chimeric or mutantmonoclonal antibodies from mouse, rat, hamster, rabbit or other species,bearing human constant and/or variable region domains or specificchanges. Techniques for generating a so-called “humanized” antibody arewell known to those of skill in the art.

A number of methods have been described to produce humanized antibodies.Controlled rearrangement of antibody domains joined through proteindisulfide bonds to form new, artificial protein molecules or “chimeric”antibodies can be utilized (Konieczny et al., 1981; incorporated hereinby reference). Recombinant DNA technology can be used to construct genefusions between DNA sequences encoding mouse antibody variable light andheavy chain domains and human antibody light and heavy chain constantdomains (Morrison et al., 1984; incorporated herein by reference).

DNA sequences encoding antigen binding portions or complementaritydetermining regions (CDR's) of murine monoclonal antibodies can begrafted by molecular means into DNA sequences encoding frameworks ofhuman antibody heavy and light chains (Jones et al., 1986; Riechmann etal., 1988; each incorporated herein by reference). Expressed recombinantproducts are called “reshaped” or humanized antibodies, and comprise theframework of a human antibody light or heavy chain and antigenrecognition portions, CDR's, of a murine monoclonal antibody.

One method for producing humanized antibodies is described in U.S. Pat.No. 5,639,641, incorporated herein by reference. A similar method forthe production of humanized antibodies is described in U.S. Pat. Nos.5,693,762; 5,693,761; 5,585,089; and 5,530,101, each incorporated hereinby reference. These methods involve producing humanized immunoglobulinshaving one or more complementarity determining regions (CDR's) andpossible additional amino acids from a donor immunoglobulin and aframework region from an accepting human immunoglobulin. Each humanizedimmunoglobulin chain usually comprises, in addition to CDR's, aminoacids from the donor immunoglobulin framework that are capable ofinteracting with CDR's to effect binding affinity, such as one or moreamino acids that are immediately adjacent to a CDR in the donorimmunoglobulin or those within about 3A as predicted by molecularmodeling. Heavy and light chains may each be designed by using any one,any combination, or all of various position criteria described in U.S.Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and 5,530,101, eachincorporated herein by reference. When combined into an intact antibody,humanized immunoglobulins are substantially non-immunogenic in humansand retain substantially the same affinity as the donor immunoglobulinto the original antigen.

An additional method for producing humanized antibodies is described inU.S. Pat. Nos. 5,565,332 and 5,733,743, each incorporated herein byreference. This method combines the concept of humanizing antibodieswith the phagemid libraries described herein. In a general sense, themethod utilizes sequences from the antigen binding site of an antibodyor population of antibodies directed against an antigen of interest.Thus for a single rodent antibody, sequences comprising part of theantigen binding site of the antibody may be combined with diverserepertoires of sequences of human antibodies that can, in combination,create a complete antigen binding site.

Antigen binding sites created by this process differ from those createdby CDR grafting, in that only the portion of sequence of the originalrodent antibody is likely to make contacts with antigen in a similarmanner. Selected human sequences are likely to differ in sequence andmake alternative contacts with the antigen from those of the originalbinding site. However, constraints imposed by binding of the portion oforiginal sequence to antigen and shapes of the antigen and its antigenbinding sites, are likely to drive new contacts of human sequences tothe same region or epitope of the antigen. This process has thereforebeen termed “epitope imprinted selection,” or “EIS.”

Starting with an animal antibody, one process results in the selectionof antibodies that are partly human antibodies. Such antibodies may besufficiently similar in sequence to human antibodies to be used directlyin therapy or after alteration of a few key residues. In EIS,repertoires of antibody fragments can be displayed on the surface offilamentous phase and genes encoding fragments with antigen bindingactivities selected by binding of the phage to antigen.

Yet additional methods for humanizing antibodies contemplated for useare described in U.S. Pat. Nos. 5,750,078; 5,502,167; 5,705,154;5,770,403; 5,698,417; 5,693,493; 5,558,864; 4,935,496; and 4,816,567,each incorporated herein by reference.

As discussed in the above techniques, the advent of methods of molecularbiology and recombinant technology, it is now possible to produceantibodies as described herein by recombinant means and thereby generategene sequences that code for specific amino acid sequences found in thepolypeptide structure of antibodies. This has permitted the readyproduction of antibodies having sequences characteristic of inhibitoryantibodies from different species and sources, as discussed above. Inaccordance with the foregoing, the antibodies useful in the methodsdescribed herein are anti-hemagglutinin antibodies, specificallyantibodies whose specificity is toward the same epitope of hemagglutininas 4F5, 5F5, and 1E11 antibodies described herein, and include alltherapeutically active variants and antigen binding fragments thereofwhether produced by recombinant methods or by direct synthesis of theantibody polypeptides.

This document provides plants, plant cells, and plant tissues expressingantibodies that maintain pharmaceutical activity when administered to asubject in need thereof. Exemplary subjects include vertebrates (e.g.,mammals, such as humans, and veterinary subjects such as bovines,ovines, canines, and felines). In certain aspects, an edible plant orportion thereof (e.g., sprout, root) can be administered orally to asubject in a therapeutically effective amount. In some aspects one ormore influenza antibody is provided in a pharmaceutical preparation, asdescribed herein.

Therapeutic, Prophylactic, and Diagnostic Compositions

In some embodiments, HA antibodies are used for diagnostic purposes. Togive but one example, HA antibodies can be used to identify a subtype,clade, and/or strain of influenza with which a subject is infected. Insome embodiments, HA antibodies can be used to identify patientpopulations that may be responsive to particular influenza treatments.

This document provides vaccine compositions comprising a least one HAantibody, fusion thereof, and/or portion(s) thereof. In someembodiments, such compositions are intended to elicit a physiologicaleffect upon administration to a subject. A vaccine protein may havehealing curative or palliative properties against a disorder or diseaseand can be administered to ameliorate relieve, alleviate, delay onsetof, reverse or lessen symptoms or severity of a disease or disorder. Avaccine comprising an HA antibody may have prophylactic properties andcan be used to prevent or delay the onset of a disease or to lessen theseverity of such disease, disorder, or pathological condition when itdoes emerge. A physiological effect elicited by treatment of a subjectwith antigen as described herein can include an effective immuneresponse such that infection by an organism is thwarted. Considerationsfor administration of HA antibodies to a subject in need thereof arediscussed in further detail in the section below entitled“Administration.”

In general, active vaccination involves the exposure of a subject'simmune system to one or more agents that are recognized as unwanted,undesired, and/or foreign and elicit an endogenous immune response.Typically, such an immune response results in the activation ofantigen-specific naive lymphocytes that then give rise toantibody-secreting B cells or antigen-specific effector and memory Tcells or both. This approach can result in long-lived protectiveimmunity that may be boosted from time to time by renewed exposure tothe same antigenic material.

In some embodiments, a vaccine composition comprising at least one HAantibody is a subunit vaccine. In general, a subunit vaccine comprisespurified antigens rather than whole organisms. Subunit vaccines are notinfectious, so they can safely be given to immunosuppressed people, andthey are less likely to induce unfavorable immune reactions and/or otheradverse side effects. One potential disadvantage of subunit vaccines arethat the antigens may not retain their native conformation, so thatantibodies produced against the subunit may not recognize the sameprotein on the pathogen surface; and isolated protein does not stimulatethe immune system as well as a whole organism vaccine. Therefore, insome situations, it may be necessary to administer subunit vaccines inhigher doses than a whole-agent vaccine (e.g., live attenuated vaccinesor inactivated pathogen vaccines) in order to achieve the sametherapeutic effect. In contrast, whole-agent vaccines, such as vaccinesthat utilize live attenuated or inactivated pathogens, typically yield avigorous immune response, but their use has limitations. For example,live vaccine strains can sometimes cause infectious pathologies,especially when administered to immune-compromised recipients. Moreover,many pathogens, particularly viruses (such as influenza), undergocontinuous rapid mutations in their genome, which allow them to escapeimmune responses to antigenically distinct vaccine strains.

In some embodiments, subunit vaccines can comprise at least oneplant-produced HA antibody. In some embodiments, about 100 μg, about 90μg, about 80 μg, about 70 μg, about 60 μg, about 50 μg, about 40 μg,about 35 μg, about 30 μg, about 25 μg, about 20 μg, about 15 μg, about 5μg, about 4 μg, about 3 μg, about 2 μg, or about 1 μg of plant-producedHA antibody and/or immunogenic portion thereof can be used to stimulatean immune response and/or to prevent, delay the onset of, and/or provideprotection against influenza infection.

In some embodiments, this document provides subunit vaccines againstinfluenza. In some embodiments, subunit vaccines comprise an antigenthat has been at least partially purified from non-antigenic components.For example, a subunit vaccine may be an HA antibody, fusion thereof,and/or immunogenic portion thereof that is expressed in a live organism(such as a plant, virus, bacterium, yeast, mammalian cell, or egg), butis at least partially purified from the non-antigen components of thelive organism. In some embodiments, a subunit vaccine is at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or at least 99% purified from thenon-antigen components of the organism in which the antigen wasexpressed. In some embodiments, a subunit vaccine may be an HA antibody,fusion thereof, and/or immunogenic portion thereof that ischemically-synthesized.

In some embodiments, a subunit vaccine may be an HA antibody, fusionthereof, and/or immunogenic portion thereof that is expressed in a liveorganism (such as a plant, virus, bacterium, yeast, mammalian cell, oregg), but is not at least partially purified from the non-antigencomponents of the live organism. For example, a subunit vaccine may bean HA antibody, fusion thereof, and/or immunogenic portion thereof thatis expressed in a live organism that is administered directly to asubject in order to elicit an immune response. In some embodiments, asubunit vaccine may be an HA antibody, fusion thereof, and/orimmunogenic portion thereof that is expressed in a plant, as describedherein, wherein the plant material is administered directly to a subjectin order to elicit an immune response.

This document provides pharmaceutical HA antibodies, fusions thereof,and/or immunogenic portions thereof, active as subunit vaccines fortherapeutic and/or prophylactic treatment of influenza infection. Incertain embodiments, HA antibodies may be produced by plant(s) orportion(s) thereof (e.g., root, cell, sprout, cell line, or plant). Incertain embodiments, provided HA antibodies are expressed in plants,plant cells, and/or plant tissues (e.g., sprouts, sprouted seedlings,roots, root culture, clonal cells, clonal cell lines, or clonal plants),and can be used directly from plant or partially purified or purified inpreparation for pharmaceutical administration to a subject.

Also provided are plants, plant cells, and plant tissues expressing HAantibodies that maintain pharmaceutical activity when administered to asubject in need thereof. Exemplary subjects include vertebrates (e.g.,mammals such as humans, as well as veterinary subjects such as bovines,ovines, canines, and felines). In certain aspects, an edible plant orportion thereof (e.g., sprout, root) can be administered orally to asubject in a therapeutically effective amount. In some aspects one ormore HA antibodies are provided in a pharmaceutical preparation, asdescribed herein.

Where it is desirable to formulate an influenza vaccine comprising plantmaterial, it will often be desirable to have utilized a plant that isnot toxic to the relevant recipient (e.g., a human or other animal).Relevant plant tissue (e.g., cells, roots, leaves) may simply beharvested and processed according to techniques known in the art, withdue consideration to maintaining activity of the expressed product. Incertain embodiments, it is desirable to have expressed HA antibodies inan edible plant (and, specifically in edible portions of the plant) sothat the material can subsequently be eaten. For instance, where vaccineantigen is active after oral delivery (when properly formulated), it maybe desirable to produce antigen protein in an edible plant portion, andto formulate expressed HA antibody for oral delivery together with someor all of the plant material with which the protein was expressed.

Vaccine compositions can comprise one or more HA antibodies. In certainembodiments, exactly one HA antibody is included in an administeredvaccine composition. In certain embodiments, at least two HA antibodiesare included in an administered vaccine composition. In some aspects,combination vaccines may include one thermostable fusion proteincomprising an HA antibody; in some aspects, two or more thermostablefusion proteins comprising HA antibody are provided.

In some embodiments, vaccine compositions comprise exactly one HAantibody. In some embodiments, vaccine compositions comprise exactly twoHA antibodies. In some embodiments, vaccine compositions compriseexactly three HA antibodies. In some embodiments, vaccine compositionscomprise four or more (e.g., 4, 5, 6, 7, 8, 9, 10, 15, or more) HAantibodies.

In some embodiments, vaccine compositions comprise exactly one HAantibody and exactly one NA antibody (e.g., NA monoclonal antibody 2B9,described in co-pending application U.S. Ser. No. 11/707,257, filed Feb.13, 2007, published as US 2008/0124272 on May 29, 2008, entitled“INFLUENZA ANTIBODIES, COMPOSITIONS, AND RELATED METHODS,” incorporatedherein by reference). In some embodiments, vaccine compositions compriseexactly two HA antibodies and exactly two NA antibodies. In someembodiments, vaccine compositions comprise exactly three HA antibodiesand exactly three NA antibodies. In some embodiments, vaccinecompositions comprise four or more (e.g., 4, 5, 6, 7, 8, 9, 10, 15, ormore) HA antibodies and four or more (e.g., 4, 5, 6, 7, 8, 9, 10, 15, ormore) NA antibodies. In some embodiments, vaccine compositions compriseexactly one HA antibody and two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, 15, or more) NA antibodies. In some embodiments, vaccinecompositions comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,or more) HA antibodies and exactly one NA antibody.

In some embodiments, vaccine compositions comprise polytopes (i.e.,tandem fusions of two or more amino acid sequences) of two or more HAantibodies and/or immunogenic portions thereof. For example, in someembodiments, a polytope comprises exactly one HA antibody. In someembodiments, a polytope comprises exactly two HA antibodies. In someembodiments, a polytope comprises exactly three HA antibodies. In someembodiments, a polytope comprises four or more (e.g., 4, 5, 6, 7, 8, 9,10, 15, or more) HA antibodies.

Where combination vaccines are utilized, it will be understood that anycombination of HA antibodies may be used for such combinations.Compositions may include multiple HA antibodies, including multipleantigens provided herein. Furthermore, compositions may include one ormore antibodies provided herein with one or more additional antibodiesand/or other therapeutic agents. Combinations of HA antibodies includeHA antibodies derived from one or more various subtypes or strains suchthat immunization confers immune response against more than oneinfection type. Combinations of HA antibodies may include at least one,at least two, at least three, at least four or more antibodies thatrecognize HA from different influenza subtypes or strains. In somecombinations, at least two or at least three antibodies that recognizeHA from different influenza subtypes are combined in one vaccinecomposition.

Additional Vaccine Components

Vaccine compositions also can include any suitable adjuvant, which canenhance the immunogenicity of the vaccine when administered to asubject. Such adjuvant(s) include, without limitation, saponins, such asextracts of Quillaja saponaria (QS), including purified subfractions offood grade QS such as Quil A and QS21; alum; metallic salt particles(e.g., aluminum hydroxide and aluminum phosphate); mineral oil; MF59;Malp2; incomplete Freund's adjuvant; complete Freund's adjuvant;alhydrogel; 3 de-O-acylated monophosphoryl lipid A (3D-MPL); lipid A;Bortadella pertussis; Mycobacterium tuberculosis; Merck Adjuvant 65(Merck and Company, Inc., Rahway, N.J.); squalene; virosomes;oil-in-water emulsions (e.g., SBAS2); and liposome formulations (e.g.,SBAS1). Further adjuvants include immunomodulatory oligonucleotides, forexample unmethylated CpG sequences as disclosed in WO 96/02555.Combinations of different adjuvants, such as those mentionedhereinabove, are contemplated as providing an adjuvant which is apreferential stimulator of TH1 cell response. For example, QS21 can beformulated together with 3D-MPL. The ratio of QS21:3 D-MPL willtypically be in the order of 1:10 to 10:1; 1:5 to 5:1; and oftensubstantially 1:1. The desired range for optimal synergy may be 2.5:1 to1:1 3D-MPL: QS21. Doses of purified QS extracts suitable for use in ahuman vaccine formulation are from 0.01 mg to 10 mg per kilogram ofbodyweight.

It should be noted that certain thermostable proteins (e.g., lichenase)may themselves demonstrate immunoresponse potentiating activity, suchthat use of such protein whether in a fusion with an HA antibody orseparately may be considered use of an adjuvant. Thus, vaccinecompositions may further comprise one or more adjuvants. Certain vaccinecompositions may comprise two or more adjuvants. Furthermore, dependingon formulation and routes of administration, certain adjuvants may bedesired in particular formulations and/or combinations.

In certain situations, it may be desirable to prolong the effect of avaccine by slowing the absorption of one or more components of thevaccine product (e.g., protein) that is subcutaneously orintramuscularly injected. This may be accomplished by use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of product then depends upon its rateof dissolution, which in turn, may depend upon size and form.Alternatively or additionally, delayed absorption of a parenterallyadministered product is accomplished by dissolving or suspending theproduct in an oil vehicle. Injectable depot forms are made by formingmicrocapsule matrices of protein in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of product topolymer and the nature of the particular polymer employed, rate ofrelease can be controlled. Examples of biodegradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsmay be prepared by entrapping product in liposomes or microemulsions,which are compatible with body tissues. Alternative polymeric deliveryvehicles can be used for oral formulations. For example, biodegradable,biocompatible polymers such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid can beused. Antigen(s) or an immunogenic portions thereof may be formulated asmicroparticles, e.g., in combination with a polymeric delivery vehicle.

Enterally administered preparations of vaccine antigens may beintroduced in solid, semi-solid, suspension or emulsion form and may becompounded with any pharmaceutically acceptable carriers, such as water,suspending agents, and emulsifying agents. Antigens may be administeredby means of pumps or sustained-release forms, especially whenadministered as a preventive measure, so as to prevent the developmentof disease in a subject or to ameliorate or delay an already establisheddisease. Supplementary active compounds, e.g., compounds independentlyactive against the disease or clinical condition to be treated, orcompounds that enhance activity of a compound provided herein, can beincorporated into or administered with compositions. Flavorants andcoloring agents can be used.

Vaccine products, optionally together with plant tissue, areparticularly well suited for oral administration as pharmaceuticalcompositions. Oral liquid formulations can be used and may be ofparticular utility for pediatric populations. Harvested plant materialmay be processed in any of a variety of ways (e.g., air drying, freezedrying, and/or extraction), depending on the properties of the desiredtherapeutic product and its desired form. Such compositions as describedabove may be ingested orally alone or ingested together with food orfeed or a beverage. Compositions for oral administration include plants;extractions of plants, and proteins purified from infected plantsprovided as dry powders, foodstuffs, aqueous or non-aqueous solvents,suspensions, or emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oil, fish oil, andinjectable organic esters. Aqueous carriers include water, water-alcoholsolutions, emulsions or suspensions, including saline and bufferedmedial parenteral vehicles including sodium chloride solution, Ringer'sdextrose solution, dextrose plus sodium chloride solution, Ringer'ssolution containing lactose or fixed oils. Examples of dry powdersinclude any plant biomass that has been dried, for example, freezedried, air dried, or spray dried. For example, plants may be air driedby placing them in a commercial air dryer at about 120° F. until biomasscontains less than 5% moisture by weight. The dried plants may be storedfor further processing as bulk solids or further processed by grindingto a desired mesh sized powder. Alternatively or additionally,freeze-drying may be used for products that are sensitive to air-drying.Products may be freeze dried by placing them into a vacuum drier anddried frozen under a vacuum until the biomass contains less than about5% moisture by weight. Dried material can be further processed asdescribed herein.

Plant-derived material may be administered as or together with one ormore herbal preparations. Useful herbal preparations include liquid andsolid herbal preparations. Some examples of herbal preparations includetinctures, extracts (e.g., aqueous extracts, alcohol extracts),decoctions, dried preparations (e.g., air-dried, spray dried, frozen, orfreeze-dried), powders (e.g., lyophilized powder), and liquid. Herbalpreparations can be provided in any standard delivery vehicle, such as acapsule, tablet, suppository, or liquid dosage. Those skilled in the artwill appreciate the various formulations and modalities of delivery ofherbal preparations that may be applied to the present disclosure.

Pharmaceutical formulations also can comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md.,2006) discloses various excipients used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Exceptinsofar as any conventional excipient medium is incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this document.

In some embodiments, the pharmaceutically acceptable excipient is atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% pure. In some embodiments, the excipient is approved for use inhumans and for veterinary use. In some embodiments, the excipient isapproved by United States Food and Drug Administration. In someembodiments, the excipient is pharmaceutical grade. In some embodiments,the excipient meets the standards of the United States Pharmacopoeia(USP), the European Pharmacopoeia (EP), the British Pharmacopoeia,and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in the formulations. Excipientssuch as cocoa butter and suppository waxes, coloring agents, coatingagents, sweetening, flavoring, and/or perfuming agents can be present inthe composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM®), sodium lauryl sulfate, quaternary ammoniumcompounds, and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g., acacia, agar, alginic acid,sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin,gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin),colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM®[magnesium aluminum silicate]), long chain amino acid derivatives, highmolecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleylalcohol, triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN®60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate[SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g., CREMOPHOR®, polyoxyethyleneethers, (e.g., polyoxyethylene lauryl ether [BRIJ®30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, and/or combinations thereof.

Exemplary binding agents include, without limitation, starch (e.g.,cornstarch and starch paste); gelatin; sugars (e.g., sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, and mannitol); naturaland synthetic gums [e.g., acacia, sodium alginate, extract of Irishmoss, panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), andlarch arabogalactan]; alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN®II, NEOLONE™,KATHON™, and/or EUXYL®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, and/or combinationsthereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macadamia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such a CREMOPHOR®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g., starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g., carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.,glycerol), disintegrating agents (e.g., agar, calcium carbonate, potatostarch, tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g., paraffin), absorptionaccelerators (e.g., quaternary ammonium compounds), wetting agents(e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g.,kaolin and bentonite clay), and lubricants (e.g., talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate), and mixtures thereof. In the case of capsules, tablets andpills, the dosage form may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

Vaccine products, optionally together with plant tissue, areparticularly well suited for oral administration as pharmaceuticalcompositions. Oral liquid formulations can be used and may be ofparticular utility for pediatric populations. Harvested plant materialmay be processed in any of a variety of ways (e.g., air drying, freezedrying, and/or extraction), depending on the properties of the desiredtherapeutic product and its desired form. Such compositions as describedabove may be ingested orally alone or ingested together with food orfeed or a beverage. Compositions for oral administration include plants;extractions of plants, and proteins purified from infected plantsprovided as dry powders, foodstuffs, aqueous or non-aqueous solvents,suspensions, or emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oil, fish oil, andinjectable organic esters. Aqueous carriers include water, water-alcoholsolutions, emulsions or suspensions, including saline and bufferedmedial parenteral vehicles including sodium chloride solution, Ringer'sdextrose solution, dextrose plus sodium chloride solution, Ringer'ssolution containing lactose or fixed oils. Examples of dry powdersinclude any plant biomass that has been dried, for example, freezedried, air dried, or spray dried. For example, plants may be air driedby placing them in a commercial air dryer at about 120° F. until biomasscontains less than 5% moisture by weight. Dried plants may be stored forfurther processing as bulk solids or further processed by grinding to adesired mesh sized powder. Alternatively or additionally, freeze-dryingmay be used for products that are sensitive to air-drying. Products maybe freeze dried by placing them into a vacuum drier and dried frozenunder a vacuum until the biomass contains less than about 5% moisture byweight. Dried material can be further processed as described herein.

Plant-derived material may be administered as or together with one ormore herbal preparations. Useful herbal preparations include liquid andsolid herbal preparations. Some examples of herbal preparations includetinctures, extracts (e.g., aqueous extracts, alcohol extracts),decoctions, dried preparations (e.g., air-dried, spray dried, frozen, orfreeze-dried), powders (e.g., lyophilized powder), and liquid. Herbalpreparations can be provided in any standard delivery vehicle, such as acapsule, tablet, suppository, or liquid dosage. Those skilled in the artwill appreciate the various formulations and modalities of delivery ofherbal preparations that may be applied to the present disclosure.

In some methods, a plant or portion thereof expressing an HA antibody,or biomass thereof, is administered orally as medicinal food. Suchedible compositions can be consumed by eating raw if in a solid form, orby drinking if in liquid form. The plant material can be directlyingested without a prior processing step or after minimal culinarypreparation. In some embodiments, a vaccine antigen may be expressed ina sprout that can be eaten directly. For instance, vaccine antigens canbe expressed in alfalfa sprouts, mung bean sprouts, spinach leafsprouts, or lettuce leaf sprouts that can be eaten directly. In someembodiments, plant biomass may be processed and the material recoveredafter the processing step can be ingested.

Processing methods useful in accordance with the present disclosure aremethods commonly used in the food or feed industry. Final products ofsuch methods typically include a substantial amount of an expressedantigen and can be conveniently eaten or drunk. The final product may bemixed with other food or feed forms, such as salts, carriers, favorenhancers, antibiotics, and the like, and consumed in solid, semi-solid,suspension, emulsion, or liquid form. Such methods can include aconservation step, such as, e.g., pasteurization, cooking, or additionof conservation and preservation agents. Any plant may be used andprocessed to produce edible or drinkable plant matter. The amount of HAantibody in a plant-derived preparation may be tested by methodsstandard in the art, e.g., gel electrophoresis, ELISA, or western blotanalysis, using a probe or antibody specific for product. Thisdetermination may be used to standardize the amount of vaccine antigenprotein ingested. For example, the amount of vaccine antigen may bedetermined and regulated, for example, by mixing batches of producthaving different levels of product so that the quantity of material tobe drunk or eaten to ingest a single dose can be standardized. Acontained, regulatable environment should, however, minimize the need tocarry out such standardization procedures.

A vaccine protein produced in a plant cell or tissue and eaten by asubject may be preferably absorbed by the digestive system. Oneadvantage of the ingestion of plant tissue that has been only minimallyprocessed is to provide encapsulation or sequestration of the protein incells of the plant. Thus, product may receive at least some protectionfrom digestion in the upper digestive tract before reaching the gut orintestine and a higher proportion of active product would be availablefor uptake.

Dosage forms for topical and/or transdermal administration of a compoundas provided herein may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, theactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required. Additionally, this disclosurecontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms may be prepared, for example, by dissolving and/ordispensing the compound in the proper medium. Alternatively oradditionally, the rate may be controlled by either providing a ratecontrolling membrane and/or by dispersing the compound in a polymermatrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid vaccines to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes may be used in the classical mantoux method of intradermaladministration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions. Topicallyadministrable formulations may, for example, comprise from about 1% toabout 10% (w/w) active ingredient, although the concentration of theactive ingredient may be as high as the solubility limit of the activeingredient in the solvent. Formulations for topical administration mayfurther comprise one or more of the additional ingredients describedherein.

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient and which have a diameter in the range from about 0.5 nm toabout 7 nm or from about 1 nm to about 6 nm. Such compositions areconveniently in the form of dry powders for administration using adevice comprising a dry powder reservoir to which a stream of propellantmay be directed to disperse the powder and/or using a self propellingsolvent/powder dispensing container such as a device comprising theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5 nm andat least 95% of the particles by number have a diameter less than 7 nm.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nm and at least 90% of the particles by number have adiameter less than 6 nm. Dry powder compositions may include a solidfine powder diluent such as sugar and are conveniently provided in aunit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1% to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide the active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising the active ingredient, and mayconveniently be administered using any nebulization and/or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface-activeagent, and/or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration may have an average diameter inthe range from about 0.1 nm to about 200 nm.

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 μm to 500 μm. Such a formulation is administered in the mannerin which snuff is taken, i.e., by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powderand/or an aerosolized and/or atomized solution and/or suspensioncomprising the active ingredient. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 nm to about 200 nm, andmay further comprise one or more of the additional ingredients describedherein.

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis document.

In certain situations, it may be desirable to prolong the effect of avaccine by slowing the absorption of one or more components of thevaccine product (e.g., protein) that is subcutaneously orintramuscularly injected. This may be accomplished by use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of product then depends upon its rateof dissolution, which in turn, may depend upon size and form.Alternatively or additionally, delayed absorption of a parenterallyadministered product is accomplished by dissolving or suspending theproduct in an oil vehicle. Injectable depot forms are made by formingmicrocapsule matrices of protein in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of product topolymer and the nature of the particular polymer employed, rate ofrelease can be controlled. Examples of biodegradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsmay be prepared by entrapping product in liposomes or microemulsions,which are compatible with body tissues. Alternative polymeric deliveryvehicles can be used for oral formulations. For example, biodegradable,biocompatible polymers such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid, canbe used. Antigen(s) or an immunogenic portions thereof may be formulatedas microparticles, e.g., in combination with a polymeric deliveryvehicle.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005.

Administration

Among other things, this document provides subunit vaccines. In someembodiments, subunit vaccines may be administered to a subject at lowdoses in order to stimulate an immune response and/or conferprotectivity. As used herein, the term “low-dose vaccine” generallyrefers to a vaccine that is immunogenic and/or protective whenadministered to a subject at low-doses. Administration of a low-dosevaccine can comprise administration of a subunit vaccine compositioncomprising less than 100 μg of an HA antibody, fusion thereof, and/orimmunogenic portion thereof.

In some embodiments, administration of a low-dose subunit vaccinecomprises administering a subunit vaccine comprising less than about 100μg, less than about 90 μg, less than about 80 μg, less than about 70 μg,less than about 60 μg, less than about 50 μg, less than about 40 μg,less than about 35 μg, less than about 30 μg, less than about 25 μg,less than about 20 μg, less than about 15 μg, less than about 5 μg, lessthan about 4 μg, less than about 3 μg, less than about 2 μg, or lessthan about 1 μg of plant-produced HA antibody, fusion thereof, and/orimmunogenic portion thereof to a subject in need thereof. In someembodiments, the plant-produced HA antibody, fusion thereof, and/orimmunogenic portion thereof has been at least partially purified fromnon-antigenic components, as described herein. In some embodiments, theplant-produced HA antibody, fusion thereof, and/or immunogenic portionthereof has not been at least partially purified from non-antigeniccomponents, as described herein. Suitable vaccine compositions foradministration to a subject are described in further detail in thesection above, entitled “Vaccines.”

HA antibodies, fusions thereof, and/or immunogenic portions thereof,and/or pharmaceutical compositions thereof (e.g., vaccines) may beadministered using any amount and any route of administration effectivefor treatment.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular composition, its mode ofadministration, its mode of activity, and the like. HA antibodies aretypically formulated in dosage unit form for ease of administration anduniformity of dosage. It will be understood, however, that the totaldaily usage of the compositions provided herein will be decided by theattending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular subjector organism will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific HA antibody employed; the specific pharmaceutical compositionadministered; the half-life of the composition after administration; theage, body weight, general health, sex, and diet of the subject; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors, well known in the medical arts.

Pharmaceutical compositions (e.g., vaccines) may be administered by anyroute. In some embodiments, pharmaceutical compositions can beadministered by a variety of routes, including oral (PO), intravenous(IV), intramuscular (IM), intra-arterial, intramedullary, intrathecal,subcutaneous (SQ), intraventricular, transdermal, interdermal,intradermal, rectal (PR), vaginal, intraperitoneal (IP), intragastric(IG), topical (e.g., by powders, ointments, creams, gels, lotions,and/or drops), mucosal, intranasal, buccal, enteral, vitreal,sublingual; by intratracheal instillation, bronchial instillation,and/or inhalation; as an oral spray, nasal spray, and/or aerosol; and/orthrough a portal vein catheter. In general, the most appropriate routeof administration will depend upon a variety of factors, including thenature of the agent being administered (e.g., its stability in theenvironment of the gastrointestinal tract) and the condition of thesubject (e.g., whether the subject is able to tolerate a particular modeof administration).

In some embodiments, vaccines are delivered by multiple routes ofadministration (e.g., by subcutaneous injection and by intranasalinhalation). For vaccines involving two or more doses, different dosesmay be administered via different routes.

In some embodiments, vaccines are delivered by subcutaneous injection.In some embodiments, vaccines are administered by intramuscular and/orintravenous injection. In some embodiments, vaccines are delivered byintranasal inhalation.

In some embodiments, vaccines as provided herein are delivered by oraland/or mucosal routes. Oral and/or mucosal delivery has the potential toprevent infection of mucosal tissues, the primary gateway of infectionfor many pathogens. Oral and/or mucosal delivery can prime systemicimmune response. There has been considerable progress in the developmentof heterologous expression systems for oral administration of antigensthat stimulate the mucosal-immune system and can prime systemicimmunity. Previous efforts at delivery of oral vaccine however, havedemonstrated a requirement for considerable quantities of antigen inachieving efficacy. Thus, economical production of large quantities oftarget antigens is a prerequisite for creation of effective oralvaccines. Development of plants expressing antigens, includingthermostable antigens, represents a more realistic approach to suchdifficulties.

In certain embodiments, an HA antibody expressed in a plant or portionthereof is administered to a subject orally by direct administration ofa plant to a subject. In some aspects a vaccine protein expressed in aplant or portion thereof is extracted and/or purified, and used for thepreparation of a pharmaceutical composition. It may be desirable toformulate such isolated products for their intended use (e.g., as apharmaceutical agent or vaccine composition). In some embodiments, itwill be desirable to formulate products together with some or all ofplant tissues that express them.

In certain embodiments, an HA antibody expressed in a plant or portionthereof is administered to a subject orally by direct administration ofa plant to a subject. In some aspects a vaccine protein expressed in aplant or portion thereof is extracted and/or purified, and used forpreparation of a pharmaceutical composition. It may be desirable toformulate such isolated products for their intended use (e.g., as apharmaceutical agent or vaccine composition). In some embodiments, itwill be desirable to formulate products together with some or all ofplant tissues that express them.

A vaccine protein produced in a plant cell or tissue and eaten by asubject may be preferably absorbed by the digestive system. Oneadvantage of the ingestion of plant tissue that has been only minimallyprocessed is to provide encapsulation or sequestration of the protein incells of the plant. Thus, product may receive at least some protectionfrom digestion in the upper digestive tract before reaching the gut orintestine and a higher proportion of active product would be availablefor uptake.

Where it is desirable to formulate product together with plant material,it will often be desirable to have utilized a plant that is not toxic tothe relevant recipient (e.g., a human or other animal). Relevant planttissue (e.g., cells, roots, leaves) may simply be harvested andprocessed according to techniques known in the art, with dueconsideration to maintaining activity of the expressed product. Incertain embodiments, it is desirable to have expressed HA antibody in anedible plant (and, specifically in edible portions of the plant) so thatthe material can subsequently be eaten. For instance, where vaccineantigen is active after oral delivery (when properly formulated), it maybe desirable to produce antigen protein in an edible plant portion, andto formulate expressed HA antibody for oral delivery together with someor all of the plant material with which a protein was expressed.

In certain embodiments, HA antibodies as provided herein and/orpharmaceutical compositions thereof (e.g., vaccines) may be administeredat dosage levels sufficient to deliver from about 0.001 mg/kg to about100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kgto about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, orfrom about 1 mg/kg to about 25 mg/kg of subject body weight per day toobtain the desired therapeutic effect. The desired dosage may bedelivered more than three times per day, three times per day, two timesper day, once per day, every other day, every third day, every week,every two weeks, every three weeks, every four weeks, every two months,every six months, or every twelve months. In certain embodiments, thedesired dosage may be delivered using multiple administrations (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, or more administrations).

Compositions are administered in such amounts and for such time as isnecessary to achieve the desired result. In certain embodiments, a“therapeutically effective amount” of a pharmaceutical composition isthat amount effective for treating, attenuating, or preventing a diseasein a subject. Thus, the “amount effective to treat, attenuate, orprevent disease,” as used herein, refers to a nontoxic but sufficientamount of the pharmaceutical composition to treat, attenuate, or preventdisease in any subject. For example, the “therapeutically effectiveamount” can be an amount to treat, attenuate, or prevent infection(e.g., influenza infection)

It will be appreciated that HA antibodies and/or pharmaceuticalcompositions thereof can be employed in combination therapies. Theparticular combination of therapies (e.g., therapeutics or procedures)to employ in a combination regimen will take into account compatibilityof the desired therapeutics and/or procedures and the desiredtherapeutic effect to be achieved. It will be appreciated that thetherapies employed may achieve a desired effect for the same purpose(for example, HA antibodies useful for treating, preventing, and/ordelaying the onset of influenza infection may be administeredconcurrently with another agent useful for treating, preventing, and/ordelaying the onset of influenza infection), or they may achievedifferent effects (e.g., control of any adverse effects). This documentencompasses the delivery of pharmaceutical compositions in combinationwith agents that may improve their bioavailability, reduce and/or modifytheir metabolism, inhibit their excretion, and/or modify theirdistribution within the body.

Pharmaceutical compositions in accordance with the present disclosuremay be administered either alone or in combination with one or moreother therapeutic agents. By “in combination with,” it is not intendedto imply that the agents must be administered at the same time and/orformulated for delivery together, although these methods of delivery arewithin the scope of this document. Compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. In will be appreciated thattherapeutically active agents utilized in combination may beadministered together in a single composition or administered separatelyin different compositions. In general, each agent will be administeredat a dose and/or on a time schedule determined for that agent.

In general, it is expected that agents utilized in combination with beutilized at levels that do not exceed the levels at which they areutilized individually. In some embodiments, the levels utilized incombination will be lower than those utilized individually.

In certain embodiments, vaccine compositions comprising at least one HAantibody are administered in combination with other influenza vaccines.In certain embodiments, vaccine compositions comprising at least one HAantibody are administered in combination with other influenzatherapeutics. In certain embodiments, vaccine compositions comprising atleast one HA antibody are administered in combination with antiviraldrugs, such as neuraminidase inhibitors (e.g., oseltamivir [TAMIFLU®],zanamivir [RELENZAAND®] and/or M2 inhibitors (e.g., adamantane,adamantane derivatives, and rimantadine).

Kits

In one aspect, this document provides a pharmaceutical pack or kitincluding at least one HA antibody as provided herein. In certainembodiments, pharmaceutical packs or kits include live sproutedseedlings, clonal entity or plant producing an antibody or antigenbinding fragment as provided herein, or preparations, extracts, orpharmaceutical compositions containing antibody in one or morecontainers filled with optionally one or more additional ingredients ofpharmaceutical compositions as provided herein. In some embodiments,pharmaceutical packs or kits include pharmaceutical compositionscomprising purified HA antibody, in one or more containers optionallyfilled with one or more additional ingredients of pharmaceuticalcompositions. In certain embodiments, the pharmaceutical pack or kitincludes an additional approved therapeutic agent (e.g., influenzaantibody, influenza vaccine, influenza therapeutic) for use as acombination therapy. Optionally associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceutical products, which noticereflects approval by the agency of manufacture, use, or sale for humanadministration.

Kits are provided that include therapeutic reagents. As but onenon-limiting example, HA antibody can be provided as oral formulationsand administered as therapy. Alternatively or additionally, HA antibodycan be provided in an injectable formulation for administration. In oneembodiment, HA antibody can be provided in an inhalable formulation foradministration. Pharmaceutical doses or instructions therefore may beprovided in the kit for administration to an individual suffering fromor at risk for influenza infection.

The representative examples that follow are intended to help illustratethe invention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the exampleswhich follow and the references to the scientific and patent literaturecited herein. The following examples contain information,exemplification and guidance, which can be adapted to the practice ofthis invention in its various embodiments and the equivalents thereof.

EXAMPLES Example 1 Cloning, Expression, and Purification of Influenza HA

The HA sequences encoding hemagglutinin (HA) from A/Anhui/1/2005 (SEQ IDNO:22), A/Bar-headed goose/Qinghai/0510/05 (SEQ ID NO:24),A/Indonesia/5/05 (SEQ ID NO:23), and A/Wyoming/3/03 (H3N2) (SEQ IDNO:34) were optimized for expression in plants and synthesized byGENEART AG (Regensburg, Germany). The PR-1a signal peptide was added tothe N-terminus and the endoplasmic reticulum retention signal (KDEL) anda poly-histidine affinity purification tag (His₆) were added to theC-terminus. The resulting sequence was inserted into the launch vectorpGRD4 to obtain pGRD4-HA (illustrated in FIG. 1). The pGRD4 vector isbased on Tobacco mosaic virus (TMV) and was engineered using thepGreen/pSoup system as a binary expression vector by introducing theCauliflower mosaic virus (CaMV) 35S promoter, the nos terminator, andthe hammerhead ribozyme sequence from the launch vector pBID4. pGRD4-HAand pSoup, which provides replication functions in trans, were thenintroduced into Agrobacterium tumefaciens strain GV3101. The resultingbacterial strain was grown in AB medium (18.7 mM NH₄Cl, 2.5 mM MgSO₄, 2mM KCl, 0.07 mM CaCl₂, 2.7 μM FeSO₄, 17.2 mM K₂HPO₄, 6.4 mM NaH₂PO₄,0.2% glucose) overnight at 28° C. The bacteria were introduced into theaerial parts of 6-week-old Nicotiana benthamiana plants grownhydroponically in rockwool slabs, by vacuum infiltration at a celldensity of OD₆₀₀=0.5. Seven days after vacuum infiltration, leaf tissuewas harvested, and homogenized using a household blender. The extractswere clarified by centrifugation (78 000×g for 30 min) and HA waspurified using Ni-column chromatography (pre-packed His Trap HP Nicolumns, GE Healthcare, NJ). Further purification was carried out byanion exchange chromatography (Sepharose Q columns, GE Healthcare, NJ)on a Bio-Rad Duo Flow system using Biologics software.

Example 2 Generation of Plants and Antigen Production

Agrobacterium Infiltration of Plants: Agrobacterium-mediated transientexpression system achieved by Agrobacterium infiltration was utilized(Turpen et al. (1993) J. Virol. Methods 42:227). Healthy leaves ofNicotiana benthamiana were infiltrated with A. rhizogenes containingviral vectors engineered to express NINA.

The A. tumifaciens strain A4 (ATCC 43057; ATCC, Manassas, Va.) wastransformed with the constructs pBI-D4-PR-NA-KDEL and pBI-D4-PR-NA-VAC.Agrobacterium cultures were grown and induced as described (Kapila etal. (1997) Plant Sci. 122:101). A 2 ml starter-culture (picked from afresh colony) was grown overnight in YEB (5 g/l beef extract, 1 g/lyeast extract, 5 g/l peptone, 5 g/l sucrose, 2 mM MgSO4) with 25 μg/mlkanamycin at 28° C. The starter culture was diluted 1:500 into 500 ml ofYEB with 25 μg/ml kanamycin, 10 mM 2-4(-morpholino)ethanesulfonic acid(MES) pH 5.6, 2 mM additional MgSO4 and 20 μM acetosyringone. Thediluted culture was then grown overnight to an O.D.600 of ˜1.7 at 28° C.The cells were centrifuged at 3,000×g for 15 minutes and re-suspended inMMA medium (MS salts, 10 mM MES pH 5.6, 20 g/l sucrose, 200 μMacetosyringone) to an O.D.600 of 2.4, kept for 1-3 hour at roomtemperature, and used for Agrobacterium-infiltration. N. benthamianaleaves were injected with the Agrobacterium-suspension using adisposable syringe without a needle. Infiltrated leaves were harvested 6days post-infiltration. Plants were screened for the presence of targetantigen expression by immunoblot analysis.

Example 3 Production of Antigen

100 mg samples of N. benthamiana infiltrated leaf material wereharvested at 4, 5, 6 and 7 days post-infection. The fresh tissue wasanalyzed for protein expression right after being harvested or collectedat −80° C. for the preparation of subsequent crude plants extracts orfor fusion protein purification.

Fresh samples were resuspended in cold PBS 1× plus protease inhibitors(Roche) in a 1/3 w/v ratio (1 ml/0.3 g of tissue) and ground with apestle. The homogenates were boiled for 5 minutes in SDS gel loadingbuffer and then clarified by centrifugation for 5 minutes at 12,000 rpmat 4° C. The supernatants were transferred to fresh tubes, and 20 μl, 1μl, or dilutions thereof were separated by 12% SDS-PAGE and analyzed byWestern analysis using anti-His6-HA mouse polyclonal antibodies.

HA expression in N. benthamiana plants infiltrated either with A.tumefaciens containing the plasmid pBID4-HA-KDEL led to a specific bandcorresponding to the molecular weight of NA-KDEL. Quantification ofHA-KDEL expressed in crude extract was made by immunoblotting both onmanually infiltrated tissues and on vacuum-infiltrated tissues.

Purification of Antigens:

Leaves from plants infiltrated with recombinant A. tumefacienscontaining the pBID4-HA-KDEL construct were ground by homogenization.Extraction buffer with “EDTA-free” protease inhibitors (Roche) and 1%Triton X-100 was used at a ratio of 3× (w/v) and rocked for 30 minutesat 4° C. Extracts were clarified by centrifugation at 9000×g for 10minutes at 4° C. Supernatants were sequentially filtered through Miracloth, centrifuged at 20,000×g for 30 minutes at 4° C., and filteredthrough a 0.45-mm filter before chromatographic purification.

The resulting extracts were cut using ammonium sulfate precipitation.Briefly, (NH₄)₂SO₄ was added to 20% saturation, incubated on ice for 1hour, and spun down at 18,000×g for 15 minutes. Pellets were discardedand (NH₄)₂SO₄ was added slowly to 60% saturation, incubated on ice for 1hour, and spun down at 18,000×g for 15 minutes. Supernatants werediscarded and the resulting pellets were resuspended in buffer,maintained on ice for 20 minutes, and centrifuged at 18,000×g for 30minutes. Supernatants were dialyzed overnight against 10,000 volumes ofwashing buffer.

His-tagged HA-KDEL proteins were purified using Ni-NTA sepharose(“Chelating Sepharose Fast Flow Column;” Amersham) at room temperatureunder gravity. The purification was performed under non-denaturingconditions. Proteins were collected as 0.5 ml fractions that wereunified, combined with 20 mM EDTA, dialyzed against 1×PBS overnight at4° C., and analyzed by SDS-PAGE. Alternatively, fractions werecollected, unified, combined with 20 mM EDTA, dialyzed against 10 mMNaH2PO4 overnight at 4° C., and purified by anion exchangechromatography. For HA-KDEL purification, anion exchange column QSepharose Fast Flow (Amersham Pharmacia Biosciences) was used. Samplesof the HA-KDEL affinity or ion-exchange purified proteins were separatedon 12% polyacrylamide gels followed by Coomassie staining.

After dialysis, samples were analyzed by immunoblotting using the mAbα-anti-His6. The His-tag was maintained by the expressed proteins, andthe final concentration of the purified protein was determined usingGeneTools software from Syngene (Frederick, Md.).

Example 4 Western Blot and ELISA Analysis of Purified ppH5HA-I

Samples of HA, purified from infiltrated N. benthamiana leaves, wereseparated on 10% SDS-polyacrylamide gels, transferred onto apolyvinylidene fluoride membrane (Millipore, Billerica, Mass.) andblocked with 0.5% I-block (Applied Biosystems, CA). The membrane wasthen incubated with a mouse monoclonal antibody against poly-His(Roche-Applied-Science, IN) followed by horseradish peroxidase(HRP)-conjugated goat anti-mouse antibody (Jackson ImmunoResearchLaboratory Inc., PA). Proteins reacting with the anti-His antibody werevisualized using SuperSignal West Pico Chemiluminescent Substrate(Pierce, IL). The image was taken using GeneSnap software on a GeneGnomeand quantified using Gene Tools software (Syngene Bioimaging, MD).

Example 5 Derivation of a Murine Hybridoma Secreting Monoclonal Antibody

Six-week-old Balb/c mice were immunized with plant-produced HAsubcutaneously at 2-week intervals on days 0, 14, and 28. Allimmunizations included 10 μg of Quil A adjuvant (Accurate Chemical, NY).Mice were boosted intraperitoneally 3-4 days prior to spleen harvest.Spleens were teased into single cell suspensions and red blood cellswere lysed (NH₄Cl solution). Splenocytes were combined with P3 myelomacells at a 1:1 ratio. A solution of 50% PEG was added and the cells wereincubated at 37° C. for 2 hrs. After the incubation, the cell pellet wasresuspended in media containing 1×HAT and plated in 96 well plates andincubated for two weeks. HAT containing media was replaced as needed. Onday 14, the cells were feed media containing HT (not HAT) for 1 week,replacing media every-other day. At this point the hybridoma cell lineswere screened by ELISA, initially on plant-produced antigen and theninactivated virus. Positive wells were put through several rounds oflimiting dilution to isolate a single clone and screened again. At thispoint, the isotype of the antibody was determined and cell supernatantswere screened for functionality by hemagglutination inhibition assays(HI) against homologous and heterologous viruses. Antibodies fromselected clones were then purified from mouse ascites at RocklandImmunochemicals.

45 million spleen cells were fused with 5 million P3XAg8.653 murinemyeloma cells using polyethylene glycol. The resulting 50 million fusedcells were plated at 5×10⁵ cells per well in 10×96 well plates. HAT(hypoxanthine, aminopterin, and thymidine) selection followed 24 hourslater and continued until colonies arose. All immunoglobulin-secretinghybridomas were subcloned by three rounds of limiting dilution in thepresence of HAT.

Hybridomas were screened on ELISA plates for secretion of H5 HA specificimmunoglobulin. Hybridomas 1E11, 4F5, 5F5, 13B8, 1E5 and 2C7 each had ahigh specific signal.

Example 6 Characterization of mAb Specificity

The specificity of each mAb to homologous and heterologous influenzaviruses was analyzed by ELISA. Plates were coated with inactivated H5N1or H3N2 virus; antibody concentration was 125 ng/ml. The reactivity ofmAb 4F5 (A/Anhui/1/05), mAb5F5 (A/Bar-headed goose/Qinghai/1A/05 and mAb1E11 (A/Bar-headed goose/Qinghai/1A/05) against a panel of influenzastrains (iA Vietnam (H5N1); iA/Indonesia (H5N1); A/Anhui/1/05 (H5N1);A/Bar-headed goose/Qinghai/1A/05 (H5N1); and iA/Wyoming (H3N2)) is shownin FIG. 2A, FIG. 2B and FIG. 2C, respectively.

Example 7 Hemagglutination Inhibition Activity of mAbs 1E11, 5F5 and 4F5

MAbs 1E11, 5F5 and 4F5 were assayed for hemagglutination inhibitionactivity. Culture supernatants of hybridoma cells were treated withreceptor-destroying enzyme (RDE; Denka Seiken Co. Ltd., Tokyo, Japan)and an HI assay was carried out with 1% horse erythrocytes containing0.1% bovine serum albumin, as described in Noah et al. (Clin VaccineImmunol., 16(4), 558-566 2009). Briefly, two-fold serial dilution ofculture supernatant or purified monoclonal antibodies were mixed with 8HAU/50 μl of influenza virus in the V-bottom 96-well plates andincubated for 45 to 60 minutes at room temperature. Horse erythrocyteswere diluted to 1% with PBS and then added to the 96-well platescontaining antibody/serum mixture. After 30-45 minutes incubation, wellswere observed for agglutination and the HI titer of the individualsamples was determined as the reciprocal of the highest dilution whichcaused complete inhibition of hemagglutination. Sheepanti-A/Vietnam/1194/04 was used as a reference serum.

The results of this experiment are shown in FIG. 3. The values in thetable are the lowest concentration (ug/ml) that inhibitedhemagglutination activity (8HAU/50 ul) for each strain. For thereference serum, the numbers shown are endpoint titers forhemagglutination activity (8HAU/50 ul) for each strain. mAbs 1E11 and5F5 inhibited hemagglutination activity of homologous as well asheterologous viruses; mAb 4F5 demonstrated hemagglutination inhibitiononly against homologous virus.

Example 8 Analysis of Binding Activity of mAbs 13B8, 4F5, 5F5, 1E11, 1E5and 2C7

Ascites produced and purified mAbs 13B8, 4F5, 5F5, 1E11, 1E5 and 2C7were screened for binding activity against a panel of inactivated wholeviruses, purified plant-produced HAs and baculovirus produced HAs. Theresults of this experiment are shown in FIG. 4. All anti-H5 mAbs wereshown to bind multiple H5N1 virus strains from both Clade 1 and Clade 2.None of the anti-H5 mAbs bound to influenza viruses of subtypes H3N2 orH1N1.

Example 9 Hemagglutination Inhibition Activity of Anti-HA mAbs

Ascites produced and purified mAbs 13B8, 4F5, 5F5, 1E11, 1E5 and 2C7were screened for hemagglutination inhibition activity according of themethod of Example 7. The results of this analysis for the anti-H5 HAmAbs, 13B8, 4F5, 5F5, 1E11, 1E5, are shown in FIG. 5; the results ofthis analysis for the anti-H3 HA mAb, 2C7, is shown in FIG. 6.

Example 10 Evaluation of Anti-HA mAbs In Vivo

The ability of the anti-H5 HA mAb, 1E11, to protect mice from challengewith influenza virus in vivo was analyzed according to the experimentaldesign shown in FIG. 7. Female mice were challenged on Day 0 with 30 μLof 10⁶⁴ EID₅₀/mL of H5N1 Avian Influenza virus. On Days 0-2, each animalwas dosed with either 100 μL or 200 μL of mAb 2B9 (an anti-neuraminidasemAb), mAb 1E11, combination of mAb 2B9 and mAb 1E11, or DPBSintravenously into the tail vein. On Day 0, Groups 1, 3, 5, and 7 weredosed beginning approximately 1 hour after challenge, and Groups 2, 4,6, and 8 were dosed beginning approximately 6 hours after challenge.Mice were assessed for clinical signs, body weight, and body temperaturechanges throughout the study (Day 0 to Day 14). The results of thisstudy are shown in FIG. 8. Treatment of H5N1 challenged mice withmonoclonal antibodies 2B9 and/or 1E11 at 1 (FIG. 8A) or 6 (FIG. 8B)hours after challenge provided 50%-90% protection from lethality. Ingeneral, mAb 1E11 provided a higher level of protection by 20%-30%compared to the 2B9 antibody. The timing for the administration of themAb did not have a substantial impact on survival or body temperature.The timing for the administration of the mAb had only a modest impact onbody weight for groups treated with the 2B9 antibody, with animals dosed6 hours after challenge appearing more stable in body weight at studytermination, indicating a better performance of the antibody.

Example 11 Half-Life Study of mAb1E11 in mice

The stability of mAb 1E11 was measured in mice according to standardmethods. The half-life of the antibody was 8.4 days when administeredintravenously and 13.7 days when administered intramuscularly.

Example 12 Amino Acid Sequences of mAbs 1E11 and 4F5

The nucleotide sequences of the light and heavy chains of mAbs 1E11 and4F5 were determined by standard methods. Conceptual translations of eachsequence are shown in FIG. 9. The heavy chain of 1E11 has the acidsequence set forth in SEQ ID NO:74; the light chain of 1E11 has the acidsequence set forth in SEQ ID NO:75. The heavy chain of 4F5 has the acidsequence set forth in SEQ ID NO:76; the light chain of 4F5 has the acidsequence set forth in SEQ ID NO:77. Signal peptide/leader sequences areshown in italics.

Sequences of the 1E11 and 4F5 heavy and light chains without signalpeptide/leader sequences are shown in FIG. 10. The heavy chain of 1E11without the signal peptide/leader sequence has the acid sequence setforth in SEQ ID NO:78; the light chain of 1E11 without the signalpeptide/leader sequence has the acid sequence set forth in SEQ ID NO:79.The heavy chain of 4F5 without the signal peptide/leader sequence hasthe acid sequence set forth in SEQ ID NO:80; the light chain of 4F5without the signal peptide/leader sequence has the acid sequence setforth in SEQ ID NO:81.

1. An isolated monoclonal antibody that binds hemagglutinin, wherein theantibody has the ability to inhibit hemagglutination, and wherein theantibody is selected from the group consisting of: an antibodycomprising a light chain variable region amino acid sequence at least95% identical to the amino acid sequence as set forth in amino acids1-97 of SEQ ID NO:79 and a heavy chain variable region amino acidsequence at least 95% identical to the amino acid sequence as set forthin amino acids 1-115 of SEQ ID NO:78; and an antibody comprising a lightchain variable region amino acid sequence at least 95% identical to theamino acid sequence as set forth in amino acids 1-96 of SEQ ID NO:81 anda heavy chain variable region amino acid sequence at least 95% identicalto the amino acid sequence as set forth in amino acids 1-112 of SEQ IDNO:80.
 2. (canceled)
 3. The antibody of claim 1, wherein the antibodycomprises a light chain variable region amino acid sequence at least 95%identical to the amino acid sequence as set forth in amino acids 1-97 ofSEQ ID NO:79, and a heavy chain variable region amino acid sequence atleast 95% identical to the amino acid sequence as set forth in aminoacids 1-115 of SEQ ID NO:78. 4-5. (canceled)
 6. The antibody of claim 1,wherein the antibody comprises a light chain variable region amino asset forth in amino acids 1-97 of SEQ ID NO:79, and a heavy chainvariable region amino acid sequence as set forth in amino acids 1-115 ofSEQ ID NO:78.
 7. (canceled)
 8. The antibody of claim 1, wherein theantibody comprises a light chain variable region amino acid sequence atleast 95% identical to the amino acid sequence as set forth in aminoacids 1-96 of SEQ ID NO:81, and a heavy chain variable region amino acidsequence at least 95% identical to the amino acid sequence as set forthin amino acids 1-112 of SEQ ID NO:80. 9-10. (canceled)
 11. The antibodyof claim 1, wherein the antibody comprises a light chain variable regionamino as set forth in amino acids 1-96 of SEQ ID NO:81, and a heavychain variable region amino acid sequence as set forth in amino acids1-112 of SEQ ID NO:80.
 12. An antibody that binds hemagglutinin, whereinthe antibody has the ability to inhibit hemagglutination, and whereinthe antibody is selected from the group consisting of: an antibodycomprising a light chain amino acid sequence at least 95% identical tothe amino acid sequence set forth in SEQ ID NO:79 and a heavy chainamino acid sequence at least 95% identical to the amino acid sequenceset forth in SEQ ID NO:78; and an antibody comprising a light chainamino acid sequence at least 95% identical to the amino acid sequenceset forth in SEQ ID NO:81 and a heavy chain amino acid sequence at least95% identical to the amino acid sequence set forth in SEQ ID NO:80. 13.(canceled)
 14. The antibody of claim 12, wherein the antibody comprisesa light chain amino acid sequence at least 95% identical to the aminoacid sequence set forth in SEQ ID NO:79, and a heavy chain amino acidsequence at least 95% identical to the amino acid sequence set forth inSEQ ID NO:78. 15-16. (canceled)
 17. The antibody of claim 12, whereinthe antibody comprises a light chain amino acid sequence as set forth inSEQ ID NO:79 and a heavy chain amino acid sequence as set forth in SEQID NO:78.
 18. (canceled)
 19. The antibody of claim 12, wherein theantibody comprises a light chain amino acid sequence at least 95%identical to the amino acid sequence set forth in SEQ ID NO:81 and aheavy chain amino acid sequence at least 95% identical to the amino acidsequence set forth in SEQ ID NO:80. 20-21. (canceled)
 22. The antibodyof claim 12, wherein the antibody comprises a light chain amino acidsequence as set forth in SEQ ID NO:81 and a heavy chain amino acidsequence as set forth in SEQ ID NO:80.
 23. The antibody of claim 1,wherein the antibody is an scFv, Fv, Fab′, Fab, diabody, linear antibodyor F(ab′)2 antigen-binding fragment of an antibody.
 24. The antibody ofclaim 1, wherein the antibody is a CDR, univalent fragment, or a singledomain antibody.
 25. The antibody of claim 1, wherein the antibody is ahuman, humanized or part-human antibody or antigen-binding fragmentthereof.
 26. The antibody of claim 1, wherein the antibody is arecombinant antibody.
 27. The antibody of claim 1, wherein the antibodyis produced in a plant.
 28. The antibody of claim 1, wherein theantibody is operatively attached to a biological agent, an imagingagent, a detectable agent, or a diagnostic agent.
 29. The antibody ofclaim 28, wherein the antibody is operatively attached to an agent thatcleaves a substantially inactive prodrug to release a substantiallyactive drug.
 30. The antibody of claim 29, wherein the drug is ananti-influenza agent.
 31. The antibody of claim 28, wherein the antibodyis operatively attached to an anti-viral agent.
 32. (canceled)
 33. Theantibody of claim 28, wherein the antibody is operatively attached tothe biological agent as a fusion protein prepared by expressing arecombinant vector that comprises, in the same reading frame, a DNAsegment encoding the antibody operatively linked to a DNA segmentencoding the biological agent.
 34. The antibody of claim 28, wherein theantibody is operatively attached to the biological agent via abiologically releasable bond or selectively cleavable linker. 35.(canceled)
 36. The antibody of claim 28, wherein the antibody isoperatively attached to an X-ray detectable compound, a radioactive ionor a nuclear magnetic spin-resonance isotope.
 37. (canceled)
 38. Theantibody of claim 28, wherein the antibody is operatively attached tobiotin, avidin or to an enzyme that generates a colored product uponcontact with a chromogenic substrate. 39-58. (canceled)
 59. Apharmaceutical composition comprising the antibody of claim 1, and apharmaceutically acceptable carrier.
 60. The composition of claim 59,wherein the composition is formulated for parenteral or topicaladministration.
 61. The composition of claim 59, wherein the antibody isa recombinant, plant-produced antibody.
 62. The composition of claim 59,wherein the pharmaceutically acceptable composition is an encapsulatedor liposomal formulation. 63-65. (canceled)
 66. A method for determiningwhether a subject is at risk for influenza virus infection, comprisingcontacting a biological sample from the subject with an antibody ofclaim
 1. 67. (canceled)
 68. The method of claim 66 further comprising,if the antibody shows detectable binding to the biological sample,administering to the subject the antibody of claim
 1. 69-70. (canceled)